A fluorescence observation device includes an oxygen saturation calculation section, a reference region setting section, a region-of-interest setting section, a normalized fluorescence intensity calculation section, and a fluorescent image generation section. The oxygen saturation calculation section calculates the oxygen saturation of the subject for each pixel. The reference region setting section sets a reference region of the subject based on the oxygen saturation. The region-of-interest setting section sets a region of interest of the subject. The normalized fluorescence intensity calculation section calculates a normalized fluorescence intensity indicating the normalized emission intensity of the fluorescence by dividing the region-of-interest fluorescence intensity, which is calculated using the pixel value of the region of interest, by the reference fluorescence intensity calculated using the pixel value of the reference region of a fluorescent image signal obtained by imaging the subject with the fluorescence. The fluorescent image generation section generates a fluorescent image based on the normalized fluorescence intensity.