Plot
The order of events will vary depending on choices that the player has made, but the overall plot remains unchanged.
The game opens with archaeologist Hershel Layton and his young assistant Luke driving to the town of St. Mystere, summoned by a letter from Lady Dahlia, widow of Baron Augustus Reinhold. The Baron stated in his last will and testament that whoever solves the mystery of the Golden Apple will inherit his fortune, and several people have attempted and failed. The two enter the town and find that most of the population is fond of puzzles and brain teasers, which Layton and Luke are adept at solving. They see a large, haphazard tower that occupies one side of town that no one can get to; people hear strange noises emanating from it at night. Layton and Luke meet Lady Dahlia and other family members, including Simon, Gordon, and the family servants. Before they can discuss the mystery further, a loud exploding sound is heard and Dahlia's cat flees out of the door. Layton and Luke retrieve the cat and, upon returning to the mansion, discover that Simon has been murdered and the case is already under investigation by Inspector Chelmey, a renowned detective. Chelmey initially suspects the two, but then tells them to stay out of the investigation. However, Matthew, the butler of the Baron's mansion, tells Layton about a small gearwheel that he found in the room near Simon's body.
As Layton and Luke continue their search for the Golden Apple, they witness the kidnapping of one of Dahlia's servants, Ramon. A strange man stuffs Ramon into a bag; they give chase but are unable to catch him, though they do find another gearwheel similar to the one before. However, they are befuddled as Ramon is back the next day as if nothing had happened. They continue to explore the town, and check the looming tower that everyone had been telling them to stay away from but are eventually led to the town's abandoned amusement park by a young girl. As they explore the Ferris wheel, a sinister figure uses a remote to tear the wheel from its moorings, sending it rolling after Layton and Luke. They barely escape as the wheel smashes through a locked building. Exploring the wreckage, they find a key shaped similarly to the tower, and Layton gets an idea of what's going on in the village. The two return to face Chelmey, who Layton realizes is an impostor. The man reveals himself as Layton's self-proclaimed arch-enemy, Don Paolo, who is seeking the Golden Apple for himself and who tried to use the Ferris wheel to knock Layton out of the picture. Paolo escapes before Layton can capture him. Luke asks the professor who Don Paolo is and why he wants revenge. Layton knows Don Paolo's reputation as an evil scientific genius but has no idea why Don Paolo hates him, implying that the two have never met before.
With Luke in tow, Layton heads for the tower, using the key to unlock a secret wall in a dead end. Inside, they discover the man that previously had kidnapped Ramon, who is named Bruno. With Bruno's help, Layton discovers the truth: all the residents of St. Mystere are robots, created by the Baron and Bruno to challenge the wits of anyone seeking the Golden Apple, hence their shared obsession with puzzles. Simon has not died, only malfunctioned; similarly, Bruno collected Ramon in order to perform repairs. Having solved the puzzle of St. Mystere, Layton and Luke climb the tower, solving more puzzles and meeting minor characters along the way. Eventually, the pair reaches the top of the tower, and much to their surprise, find a beautifully kept cottage there. Inside, the young girl from before awaits. She reveals herself as Flora Reinhold, the only daughter of the Baron. She is the "Golden Apple" that the robots were protecting until she became an adult.
Layton's triumph is short-lived as Don Paolo returns in a flying machine and starts demolishing the tower. Luke escapes down the stairs, but Layton is forced to improvise a glider to take Flora and himself to safety as the tower collapses. Don Paolo, with his machine malfunctioning, drops a bag containing the stolen Simon. The villain swears revenge and leaves, and the three reach the town safely. As Flora hugs the Professor and laughs, an apple-like birthmark can be seen on her shoulder. As they regroup at the Reinhold manor, Layton realizes that there is more to the treasure than just Flora, as the birthmark points to the Baron's riches. Luke finds a switch on the portrait of Flora in the same location as her birthmark which leads to a secret room filled with gold.
A voice recording from the Baron, intended for those who solved the mystery, congratulates Layton. The voice tells Flora to take the treasure, explaining that if it is taken, all the robots will stop functioning. Flora opts to leave it as a way to repay the robots for their services in protecting her and her new friends. As the game ends, Layton, Luke, and Flora leave St. Mystere without the treasure, allowing the residents to continue on with their lives. The three (and other characters) are shown laughing and living together during the game credits.
As the first part of a trilogy, the main story ends with a "to be continued" message with a picture of Luke and Layton at a train station, hinting at the plot of Professor Layton and the Diabolical Box.