The two sisters, Ai and Yuuko, have taken different paths in life. Yuuko has been taken away by a mysterious gang. Ai is determined to save her from the clutches of crime. In a seemingly all-female city overrun by insane workers, biker gangs, and assorted criminals, Ai and her friends Chika and Makoto decide to work together in order to restore the world to normality again.

Ane-san is what is commonly called beat-'em-up, i.e. a fighting game that allows limited movement through side-scrolling environment. The player can choose to control a variety of female fighters. In the beginning, only Ai, Chika, and Makoto are selectable. Ai is the all-around balanced character, Chika is fast and fragile, while Makoto is very strong, but slow. Later in the game, other playable characters become available. Each fighter has a health points bar, which can be restored by healing items scattered through the levels. Once a character is dead, the player can continue playing with another one.