George is a fourth grader at Jerome Horwitz Elementary School who possesses a strong imagination and a love for pulling pranks. He spends his afternoons in his backyard treehouse writing stories for homemade comic books, which he then sells on the school playground. Though his practical jokes frequently ruin school events and anger the administration, he maintains a loyal, carefree attitude alongside his creative partner.

Harold is a fourth-grade student at Jerome Horwitz Elementary School who operates as the artistic half of his creative duo with George. He spends most of his free time in a treehouse, illustrating the comic books that his friend writes. He shares a deep enthusiasm for harmless trouble and eagerly participates in elaborate school-wide practical jokes.

Mr. Krupp is the ill-tempered principal of Jerome Horwitz Elementary School. He openly despises children, laughter, and the sounds of students playing at recess. He holds a special hatred for George and Harold's homemade comic books and dedicates his time to finding ways to permanently discipline the two boys, eventually resorting to blackmail after catching them on videotape.

Captain Underpants is an eccentric superhero characterized by his unusual crime-fighting costume, which consists entirely of white underwear and a red cape. He fights for truth, justice, and all things pre-shrunk and cottony. Initially a fictional creation of two fourth graders, his identity takes on a bizarre real-world life of its own following a hypnotic mishap.

Doctor Diaper is a tiny, diabolical supervillain who wears nothing but a large baby diaper. He operates out of a hidden warehouse and plans to destroy the moon with a laser machine in order to trigger natural disasters and take over the Earth. Despite his grand schemes for global domination, he is highly sensitive and easily distracted by personal embarrassment.