Splinter was the pet rat of Hamato Yoshi in Japan who followed the teachings of his owner from his cage. When forced to America and after his master's death, Splinter found himself in the sewers of New York where he found four turtles. One morning Splinter awoke to find the turtles playing in a radiocative ooze spilling out of the steel drum. Soon, he noticed that not only were the turtles changing, so was he. Eventually, he grew to his fully mutated form and began to teach his adoptive sons the ways of honor and the path of the ninja.
- Teenage Mutant Ninja Turtles - A team of anthropomorphic freshwater turtles who are the protagonists of the series:
- Leonardo - "Leo" is a hardworking ninjutsu student, learning how to be an effective leader for his three often unruly brothers. He wears a blue mask and fights with two katana swords called Niten Ryu.
- Donatello - "Donnie" is in charge of the design and manufacture of all of the tools and weapons in the Turtles' arsenal, and he is also a gifted hacker. He wears a purple mask and fights with a rokushakubo which converts to a naginata. He has a crush on April in this series and his brothers tease him about it.
- Raphael - "Raph" often lets his impulsiveness get in the way of rational thinking. He wears a red mask and fights with two sai.
- Michelangelo - "Mikey" loves video games, skateboarding, pranks and pizza. He wears an orange mask and fights with two nunchaku, which convert into kusarigama. Though he possesses a child-like innocence, Mikey always brings his best game to a fight.
- April O'Neil - The 16 year-old granddaughter of Doctor Amelia O'Neil who likes to play video games and eat pizza.
- Splinter - Splinter is the mutant rat leader of the entire family, and serves as a father figure for the Turtles. He once was a pet for a renowned martial artist, Hamato Yoshi in Japan. It was from watching Yoshi that Splinter first learned the art of ninjitsu which he then passes on to the Turtles. Splinter often acts as much as a spiritual guide and counselor to the team as he does their teacher. While he does not wield a traditional Japanese weapon, Splinter carries a cane which he can utilize as a weapon (like a bo staff) with fierce proficiency. Unlike the previous animated series, the Turtles often refer to Splinter as their father, rather than their master.
- Casey Jones - He is a confidant and friend to the Turtle family. He operates as a vigilante, at first on his own, and then later as a member of the Turtles' extended crew. While at first being more closely aligned with Raphael, Casey quickly becomes part of the family and valuable fighter. When in his full get-up he wears a hockey mask, and utilizes a hockey stick and other sports paraphernalia as his weapons. He takes to April quite quickly, and although she dislikes him initially the two become romantically involved.
- Russell Hardwell - He is a partner and friend to the Turtle family and Casey Jones. He is a member of the Turtles' extended crew. He speaks in slang and has an encyclopedic knowledge of the game. His intense opinions, his appearance and his unorthodox style of battle and the way he expresses himself all lend themselves to comedy.
- Foot Clan - A ninja organization that is led by the Shredder.
- The Shredder - One of the primary antagonists of the series and leader of the Foot Clan.
- Karai - A rebellious teen member of the Foot Clan and an expert martial artist who stirs up trouble for the Turtles.
- Hun - Shredder's muscle, and leader of the Purple Dragons, an urban street gang that Shredder sometimes utilizes. He constantly seeks the Shredder's approval and leads the Foot into battle against the Turtles. He is revealed to have been responsible for the death of Casey Jones's father years earlier, and as such, is a particular enemy of Casey.
- Sharkfin - Bill Dundee is an Australian martial artist and secretly a member of the Foot Clan. He is ordered to New York with El Panthero to find Splinter. As Dundee was previously bitten by the great white shark, the mutagen transforms him into Sharkfin, a shark-like mutant with big arms and legs. After his transformation, he breathes both air and water.
- El Panthero - Mona Cantino is a Spainish art dealer until Shredder brought into the Foot Clan. Shredder orders Mona Cantino to assist Bill Dundee in finding Splinter. As Mona Cantino was previously scratched by a cougar, the mutagen transforms her into a cat-like mutant El Panthero.
- Armordillo - Snively is a large fat man that works for the Shredder and becomes a member of the Foot Clan. As Snively previously touched a giant armadillo, the mutagen transforms him into Armordillo, an hunchbacked, monstrous, armadillo-like mutant with large claws.
- Foot Ninjas - The foot soldiers of the Foot Clan.
- The Krank - One of the main antagonists of the series. The Krank are a race of brain-shaped aliens that pilot robotic bodies and who are responsible for bringing mutagen to Earth.
- Purple Dragons - A street gang that has been extorting shop owners for protection money. The Purple Dragons have become allies with the Foot Clan and were responsible for stealing April's cell phone.
- Baxter Stockman - Baxter Stockman is a technical genius and the scientist responsible for creating the mousers: a series of mouse-like robots that can eat through practically anything. While at first it appears the robots were created in order to help control the cities growing rat problem, it is revealed that Stockman created them as a technical resource for the Shredder. Following the incident with the M.O.U.S.E.R.S., Hun and Sharkfin bring Stockman to Shredder. Shredder threatened to kill Stockman for interfering with his plan to kill the Turtles, but spares him and comments on how his intelligence could be useful.
- M.O.U.S.E.R.S. - Mobile Offensive Underground Search Excavation and Retrieval Sentries (M.O.U.S.E.R.S.) are small robots that were created by Baxter Stockman to steal on his behalf. They attack anything tagged with a certain unnamed radioisotope.
- Triceratons - They are a warring alien race with the appearance of a humanoid triceratops that the Turtles come into contact with when they are accidentally transported across several galaxies at the end of the first season while attempting to find the missing Master Splinter. The Turtles then become a public enemy of the Triceratons for harboring a fugitive known as the Fugitoid, who possesses a transporting technology that the Triceratons desperately want. The Triceratons wish to use Professor Honeycutt's technology to aid them in their war with the Federation. Later in the series, the Triceratons invade earth believing that the Fugitoid is still in hiding there when in reality he has gone to live with the Utrom on their homeworld.
- Lord Hornix - Prime Lord Hornix of the Triceraton Republic led the war against the Federation of the planet M'ortex.
- Commander Moxor - The military commander of the Triceraton Republic, Moxor is the counterpart of Colonel Daniel and Lieutenant Zuno when it comes to military matters. Moxor is the dedicated right hand of Prime Lord Hornix.
- Federation - Federation is a military organization of humans that the Turtles meet when they are in space. The Federation is locked in a war with the Triceratons and both sides seek to gain access to a robot known as the Fugitoid. Professor Honeycutt (the Fugitoid), having created a dimensional portal that would give either side a huge tactical advantage in their current battles, has become the prized object of the war. When the Turtles befriend Professor Honeycutt, the Federation seeks to destroy them for harboring him.
- Colonel Daniel - Colonel Daniel is an intimidating, tyrannical, vehement and ruthless leader of the Federation and will use any means necessary to win.
- Lieutenant Zuno - The militay commander of the Federation. Zuno is the lieutenant of Colonel Daniel.
- Hong - Hong is Colonel Daniel's miserable underling and assistant, as well as his nephew. He is a double agent for the Triceratons, revealing Daniel's plans, as long as Commander Moxor has the drug Xemorene he craves. He is arrested when Moxor reveals that Daniel's “trusted” aide, Hong, has been working as an agent for the Triceratons.
- Mold Mutant - Klay is a thug working for the Krank as their gateway driver. The Turtles interrogate him and find out where the Krank are holding April and Amelia O'Neil. During their mission to rescue the O'Neils, the Turtles crash a truck full of mutagen into the Krank base. Klay is exposed to the the mutagen and some nearby green slime molds which turns him into a skeletal monster covered in a green slime that Michelangelo dubs "Mold Mutant". The Turtles fight Mold Mutant. Leonardo lures him into a sewers where Mold Mutant falls down. After the Turtles leave, Mold Mutant is now furious that he has been transformed into a slime monster, the one thing he hates most in the world, even more than the Shredder. Consumed with hatred, he joins the Shredder to help him destroy the Turtles.
- Rat King - Dr. John Shakespeare is a scientist who was working for the Kraang on an experiment involving mutagen, biomolecule and fox DNA. Believing that he was close to developing a biomolecule that could make him psychic, Shakespeare tested the hybrid molecule on his colleague Dr. Knockwell transforming her into a woman/fox mutant. After the Turtles figured out that Shakespeare was lying about Knockwell's involvement in the research, they confronted him. Shakespeare injected himself with the molecule, making himself psychic and able to predict the Turtles' every move. Donatello figured out how to close off his mind from Shakespeare to defeat him. Shakespeare escaped after the Turtles could interrogate him about the Krank's plot, but before he gets mutated accidentally, turning him into a mutant rat. In "I, Monster", Shakespeare is now furious that he has been transformed into a giant rat, and he recreates a psychic biomolecule. An accident in the lab gives him psychic control over all the rats in the city including Splinter. The Rat King manipulates Splinter into fighting the Turtles, but Splinter frees himself from mind control and defeats the Rat King.
- Dr. Ameila O'Neil - A scientist who is April O'Neil's grandmother. She and April were captured by the Krank. Although April was rescued by the Turtles, the Krank made off with Ameila.
- Utroms - Utroms are the peaceful aliens who resemble a brain.
- Mr. Mortu - Mortu was the captain of the Utrom ship that crashed on Earth. After the crash, he remained the field-leader of the marooned Utroms, and worked as their liaison to the Humans. In the modern day, this makes him CEO of the front company TCRI. His name is "Utrom" backwards.
- Dr. Xeinos - She was a beautiful, Utrom scientist who found and raised Leatherhead as her son.
- Leatherhead - Leatherhead is an alligator that was originally owned by a young boy until he died in a car accident and Leatherhead fall into a storm drain, where he was found by Dr. Xeinos and other Utroms when they discovered that their mutagen (the same that transformed the Turtles) made him sentient. Leatherhead lived with the Utroms peacefully until the Krank took him to their dimension, and experimented on him. Leatherhead eventually escapes from the Krank.
- Andrea Birdwell - A 16-year-old, African American girl who is April's best friend and Leonardo's love interest, sweatheart, and girlfriend. She mostly wears a white sleeveless shirt and pink skirt, and white boots. Andrea befriends the Turtles after the Krank kidnap her. After the Krank took her, they turned her into a white, cat-like mutant, and experimented on her. She is considered a slight damsel-in-distress, as she has been kidnapped by the enemies She truly belongs with Leo as his true love.
- Zog - One of the Triceraton soldiers left on Earth when the Utroms left. Some time later, Raphael found him in the sewers, and the Turtles used him to aid in their attack on the Krank and later the Foot Clan.
- Traximus - A noble warrior of the Triceraton Republic who believed that its old virtues. Like the 2003 incarnation, he is a friend of the Turtles.
- The Fugitoid (A.K.A. Professor Honeycut) - A robot from the distant galaxy. Originally an alien with an appearance no different from humans and living on the planet M'ortex, he was forced to take his current form after he was struck by lighting during a storm which transferred his consciousness into his worker robot. He invented a transmatter portal device, which gives the user the ability to teleport anywhere in space. Two warring factions, the Federation and the Triceretons, seek to gain control of the device. This makes the professor a fugitive on the run. When the Turtles encounter him, they attempt to help him escape his fate and keep the device away from both factions.
- Dr. Tamara Knockwell - Scientists Tamara Knockwell and her partner Dr. John Shakespeare were approached by the Krank to work on an experiment involving mutagen, biomolecule and fox DNA. April, Russell and the Turtles learn Knockwell has gone missing and investigate, to discover Shakespeare had transformed Knockwell into a fox-like mutant.