A fluorescence observation apparatus according to the present invention includes a light source section that emits excitation light for exciting a first fluorescent substance and a second fluorescent substance, a fluorescence detection section that detects first fluorescent light emitted when the first fluorescent substance is excited and second fluorescent light emitted when the second fluorescent substance is excited, a correction value calculation section that calculates a correction value for canceling a color mixture between the first fluorescent light and the second fluorescent light based on feature values corresponding to characteristics of the first fluorescent substance and the second fluorescent substance, a detected image generation section that generates a first detected image corresponding to a detection result of the first fluorescent light and a second detected image corresponding to a detection result of the second fluorescent light, and an image correction section that corrects a luminance value of the first detected image to a luminance value corresponding to intensity of the first fluorescent light and corrects a luminance value of the second detected image to a luminance value corresponding to intensity of the second fluorescent light.