The events of this game take place one year after Professor Layton and the Strange Village.

Professor Hershel Layton and his apprentice, Luke, receive a letter from Andrew Schrader, respected scholar, mentor and friend of the professor, which contains information about the celestial box, commonly known as "Pandora's Box". Worried, Layton and Luke decide to go see Andrew, but once there they find him unconscious and the box has disappeared. In the scientist's office, Layton and Luke find a ticket for the Molentary Express and a torn photo. They decide to take the train to find Pandora's Box. Once they arrive in Dropstone, the two heroes discover a mystery destination on the Molentary Express: Folsense, a ghost town.