- Yumi Yoshimura, the younger of the duo, is depicted as a cynical, sarcastic punk rocker with lavender hair and blue eyes. She dresses in a heavy metal/grunge/goth style with studded necklaces and bracelets, a light purple T-shirt with a skull that has the same facial expressions as she does and seems to have a mind of its own, a black miniskirt, purple bike shorts, and black army boots. When she, along with Ami, is competing with villains, Yumi magically transforms her outfit into a battle uniform, which she wears a dark purple tanktop with a black horizontal stripe in the middle, white pantyhose and black Mary Jane shoes. Her main signature color is purple. She is the most legendary, hard-rocking, guitar-griding goddess with an absolutely infallible sense of cool, who is born several hundred million years ago and preaching the gospel while rocking hard. Her sarcastic wit and deadpan delivery are the perfect compliment to Ami's wide-eyed, unbridled enthusiasm. Being younger, Yumi is also more impetuous than Ami and rushes headlong into a decision before thinking, however, Yumi has a mature side, too, and sometimes reasons with Ami's childish obsessions, although she herself is two years younger. Yumi loves things like farting, ninjas, monster trucks, fighting, car crushers, R-rated action movies, and anything else rough and tough. Yumi is considered the biggest name in rock history. Yumi is voiced by Grey DeLisle.
- Ami Onuki the older one of the duo is depicted as a peppy, optimistic, and cute girl with rose hair and eyes. She favors bubblegum pop and wears a 1960's go-go dress, also sporting a flower in her hair and white knee-high go-go boots. When she, along with Yumi, is competing with villains, Ami magically transforms her outfit into a battle uniform, which she wears a yellow tanktop with a black horizontal stripe in the middle, white pantyhose and black Mary Jane shoes. As the optimist, Ami always looks on the bright side of things and finds a solution to every situation (being two years older and more mature). Ami is voiced by Janice Kawaye.
- Kaz Harada is the duo's well-intentioned yet money-grubbing manager, whose successes are usually balanced out by his foolish decisions. His appearance and name are based on Puffy AmiYumi's real-life manager, Kaz Harada. Veteran character actor Keone Young provides his voice. He is also a huge fan of sumo wrestling, as seen in the episode "Sumo Kaz".
- Jang Keng and Tekirai are Yumi's and Ami's pet cats, respectively. Jang Keng (sometimes called "Jengo") is a black cat and Tekirai ("Teki" for short) is a fluffy white cat. The girls adore their cats, but Kaz is a constant victim of their mischief. Janice Kawaye voices Tekirai while Grey DeLisle does Jang Keng.
- Harmony' 'is the self-proclaimed "Number One Fan" of the girls and of Kaz by the end of the episode "Dis-Harmony" and in "Fan Clubs". She is a six-year-old psycho girl who is so obsessed with the duo that she follows it around the world, annoying Ami and Yumi to no end. At the end of her first appearance, she becomes obsessed with Kaz's singing and this becomes a running gag in some episodes, appearing from nowhere and telling Kaz she is his #1 fan. The character is voiced by Sandy Fox.
- Eldwin Blair is a sinister land developer, and the antagonist in the two episodes in which he appears. He usually tries to tear down beloved places for his own selfish needs.
- King Chad is "a bad boy super hunk", as once described by Ami and Yumi. He is obsessed with the card game Stu-Pi-Doh! (a parody of Yu-Gi-Oh!) and thinks highly of himself. In "Janice Jealous", he had a girlfriend named Janice who shared his interests in video games, yo-yo tricks, and comics. True to his name, King Chad wears a fur-lined cape and gold crown, and sits on a beautifully-adorned throne. In the episode "Janice Jealous", the character King Chad goes out with is based on Janice Kawaye, Ami's voice actress. She also voiced the character Janice. Chad is voiced by Katie Leigh.
- 'Wall' is a hulking yet slow-witted man hired by Kaz as the girls' bodyguard. He is overprotective, pummeling anyone with whom Ami and Yumi attempt to make contact. Kaz later hired him as a bouncer for a party he was throwing and, more recently, Ami had him try being a roadie. He is voiced by Will Ryan.
- Domo'is Kaz's dog. He looks much like his owner, even sharing a similar style of glasses (Kaz is the only one who doesn't see the resemblance). Like Kaz, he is constantly in conflict with the cats, especially since he enjoys eating their food.
- Talent Suckers are a vampire rock trio from Transylvania who first appeared in the eponymous episode, "sucking" Ami and Yumi's talent from them. They returned in two other episodes. The straight man of the trio is Vlad (Nathan Carlson), a tall vampire who speaks with a Transylvanian accent. The second band member is a short vampire, Nicolai (Corey Burton), who is easily made nervous. (A running gag has Vlad slapping Nicolai back to his senses.) The third Talent Sucker is Mitch (also voiced by Nathan Carlson), a vampire who does not talk, but instead grunts and groans.
- Atchan is a character Ami and Yumi met at Camp Youwannasushi. He thinks he is a superhero. He is based on Vo Atsushi, the lead singer of the J-Pop band New Rote'ka. Atchan constantly refers to himself in the 3rd person. Before becoming a superhero, Atchan's face resembles Mac from Foster's Home for Imaginary Friends'. After he declares himself a hero, his looks change to look more like Kamen Rider's. He has a painted star over his right eye resembling that of Kiss (band) guitarist Paul Stanley. He is voiced by Rob Paulsen.
- Julie is Yumi's friend in the episode "Julie AmiYumi", which was not aired in the United States. She is a former band member who's signature color is green and she plays a keyboard guitar. She treats Yumi kindly, but is always mean to Ami. In the episode, she tries to take out Ami and Yumi as revenge for them stealing the stardom which she believed to be rightfully hers. She could not remember Ami's name and called her Arthur. At the end when she is standing in the rain she swore revenge. She is voiced by Lara Jill Miller .