During one of the many monster attacks on the city where the story takes place, Silk Koharuno and her two friends attempt to get closer to the monster to get a better view. Unfortunately, the city's protector, UFO-Man (an Ultraman Parody), comes to the rescue but inadvertently steps on them and crushes the girls to death. Feeling sorry for what he had done, UFO-Man blames their deaths on the monster, Gullmark, and promises to bring them back to life by lending them his power. However, now they must become the city's protectors as well, and so the Ultimate Girls are born.
- Silk Koharuno - The main heroine of the trio. Although very shy and reserved about her new duty, she is the first of the three to demonstrate their new powers as UFO-man's successor, growing into a giant heroine. Only minutes after the transformation, she discovers her unusual battle suit is deteriorating little by little, until she is totally nude. This is, in fact, the source of her power as Ultimate Girl, since the most embarrased she becomes, the stronger her powers are. Being the most shy of all three girls, she is the most powerful of them all. She is referred to by the crowd as Ultimate Girl Hinnyu (Little Breasts).
- Vivenne Ōtori - One of Silk's best friends. She along with Silk and Tsubomi were accidentally crushed and resurrected by UFO-man. Nervous about indecent exposure, she does not try her powers as an Ultimate Girl until Tsubomi fails in her first transformation and Silk refuses to do so again. Left no choice, she offers to go, being dubbed Ultimate Girl Kyonyū (Big Breasts) by spectators. When attempting to avoid exposing herself when her suit deteriorates, Vivienne is the first to learn how their powers are supposed to work, by being embarrassed, they generate an energy which can be projected as a weapon against the giant monsters. She has a secret crush on Silk.
- Tsubomi Moroboshi - Silk and Vivienne's friend and classmate. Tsubomi is a cosplayer by hobby, so when she realized that she too was a giant heroine, she took the idea without feeling any shame or embarrassment. However, because of this attitude, she was basically useless, since the source of their strength comes from being thoroughly embarrassed and projecting that emotion into an energy that can defeat their giant adversaries. Even after learning this, Tsubomi is not fazed and enjoys her new profession as a heroine and still manages to hold back the many giant monsters they encounter by tricking them instead of using brute power. It was Tsubomi's idea to go by the name "Ultimate Girl." She is called Ultimate Girl Loli (from the term Lolita) by the spectators, because of her oveall little girl appearance, even if she's the same age as Silk and Vivenne.
- UFO-Man - The giant hero who has always been fighting innumerous giant extraterrestrial monsters that rampage throughout Japan, until the he accidentally steps on Silk, Vivienne, and Tsubomi. Out of sympathy for the girls (while also blaming the monster for their deaths), he sacrifices much of his power and energy to resurrect the three of them. In doing so, his body shrinks considerably, forcing him to rely on a UFO-like craft to levitate. By giving the girls his power, they are forcibly pressed into serving as giant heroines in his stead, by changing his hover craft into a wand which they must grab in order to invoke the transformation process. Only one of the girls can transform into Ultimate Girl at the same time, since UFO-man can't give power to the three of them at once. UFO-man is a parody of Ultraman.
- Yosaku Okamura - A journalist who follows the UFO-man and Ultimate Girls story. He offered a reward for photos showing who the Ultimate Girls really were. He is the one that gives the three Ultimate Girls their fan names (Hinnyu, Kyonyū and Loli).
- Makoto Moroboshi - Tsubomi's older brother and a member of the high school press club as a photographer. He is always looking for photos of UFO-man, and is pursuing the reward offered for taking photos of the Ultimate Girls.
- Mayu Koharuno - Silk's older sister and a reporter working with Okamura.
The monsters of the series are born by green alien slime fusing to human beings with the monsters representing their personalities. If the opal-like cores are destroyed or separated the monster will disintegrate into gold particles of light.
- Gullmark - Appears in episode 1. Its only known power is having a highly articulate body. It is a homage to Gomess from Ultra Q.
- Shupo - Appears in episodes 2 and 3. Its only known power is morphing into a steam engine train. It is a homage to King Joe from Ultra Seven.
- Ochuusha - Appears in episode 4. Powers include a syringe on arms, super speed, and stethoscope on the pelvis. It is a homage to Alien Baltan from the original Ultraman with characteristics of Zetton (also from Ultraman) and Alien Metron (from Ultraseven).
- Megami Mask - Appears in episode 5. Powers include a pair of glue bazookas on the back, wrestling skills, and summonable steel armor. He is a reference to the editor of Megami Magazine, the magazine that publishes the Ultimate Girls manga and his mask is arguably a reference to Ultimate Muscle.
- Hachiro - Appears in episode 6. His only known power is a boxing glove on him main pair of tentacles.
- Mahler - Appears in episode 7. Powers include burrowing, emitting hypnotic waves from the giant metronome on its face, and a maestro wand. It is a homage to Mochiron from Ultraman Taro.
- Dainioh - Appears in episodes 8 and 9. Powers include a club, a constricting bead necklace, a spare body, burrowing by spinning, swimming, and six extra arms in its body. It is a homage Majin from Daimajin.
- Mushuusaa - Appears in episode 10. Powers include a pair of scythe arms and a whip tail. It is a homage to Gyaos and Gigan.
- Giant Makato - Appears in episodes 11 and 12. He is a homage to super robots such as Mazinger Z and the guyver units from Bio Booster Armor Guyver.