Ami Matsuzaki is a five-year-old girl settling into a new home with her parents. In her bedroom, she discovers a magical doorway in the floor, connecting her to AniYoko, an unseen alternate world inhabited by sentient stuffed animal-like creatures. This doorway is the primary link between the two worlds.

Despite her age, Ami often displays maturity and intelligence, acting as the most rational participant in her encounters. She befriends three AniYoko residents: Iyo, a rabbit; Kenta, a bear; and Issa, a panda. Known as the "Happy AniYoko Trio," they frequently visit through the doorway, involving Ami in their chaotic and unpredictable activities. Though initially bewildered, annoyed, and flustered by their antics, she gradually becomes accustomed to their presence, incorporating them into her regular life.

Ami attends kindergarten with her close human friend, Kumiko Takeda (Ku-chan). Ku-chan uniquely possesses the ability to see the AniYoko Trio as they truly are, sharing in Ami's unusual experiences. Ami keeps AniYoko and its inhabitants a secret, especially from her parents, Ayako and an unnamed father, who are sometimes heard but never fully seen. Her mother occasionally notices the aftermath of strange events without knowing their source.

The AniYoko doorway transforms her bedroom environment, enabling surreal adventures without leaving the room. She and the trio are frequently whisked away to unexpected locations like jungles, forests, baseball stadiums, or RPG-style dungeons, all contained within her bedroom.

Born on December 15th (Sagittarius) with blood type B, Ami's relationship with the trio evolves from initial surprise and wariness to acceptance. She shares parts of her human life with them while navigating their disruptive influence. Kenta develops a crush on Ku-chan, adding complexity to the group dynamic. While the magical doorway offers potential access to AniYoko for Ami, the trio's often frightening stories about their world deter her from visiting.