The series chronicles the adventures of an independent, fearless and brave Thai-American girl named Anne Boonchuy (Brenda Song). On her 13th birthday, Anne is peer-pressured into stealing a mysterious music box (known as the Calamity Box) that magically transports her and her two best friends Sasha Waybright (Anna Akana) and Marcy Wu (Haley Tju) to the world of Amphibia, a wild marshland tropical island full of anthropomorphic amphibians and threatening creatures, where they are separated from each other. Anne is taken in by the Plantars, a family of frogs that consists of excitable young Sprig (Justin Felbinger); unpredictable and adventurous baby pollywog Polly (Amanda Leighton); and overprotective and traditional grandfather Hop Pop (Bill Farmer) who live on a farm in the town of Wartwood. As she bonds with her newfound family, Anne gradually learns what it means to be a hero and develop a true friendship, all the while trying to find her friends and return home. Sasha allies with Captain Grime (Troy Baker), leader of the warfaring toads of Toad Tower, who seek to control their home of Frog Valley. Marcy ends up in Newtopia, the capital of Amphibia, the seat of the government and home to the ruler of Amphibia.
In the second season, Anne and the Plantars go on a road trip to Newtopia, to learn the secrets of the Calamity Box and find a way to get Anne home. There, Anne reunites with Marcy, who offers to help restore the box's power through a series of ancient trials, unaware that Amphibia's ruler, King Andrias (Keith David), secretly has plans for them. Meanwhile, Sasha and Grime plot to invade Newtopia and overthrow the monarch, allowing the toads to rule over all of Amphibia.
In the third and final season, Anne and the Plantars are transported to Anne's home in the suburbs of East Los Angeles. Anne must now help the frog family adjust to the human world and keep their identities a secret while searching for a way to return to Amphibia and stop Andrias's invasion of the multiverse. When they return, they discover Sasha and Grime have started a resistance force in Wartwood and aid them in their rebellion against Andrias, who is using Marcy as a vessel for his master, an entity known as the Core.