A fluorescence observation apparatus according to the present invention includes a light source section that can emit a plurality of excitation light beams for exciting a plurality of fluorescent substances and a reference light, an image pickup section that picks up images of a plurality of fluorescent light beams emitted by emitting the plurality of excitation light beams to the plurality of fluorescent substances and reflected light of the reference light, an image generation section that generates an image signal corresponding to the plurality of fluorescent light beams and the reflected light of the reference light whose images are picked up by the image pickup section, a brightness detection section that detects brightness of a plurality of image signals corresponding to the plurality of fluorescent light beams and brightness of an image signal corresponding to the reflected light of the reference light, and a brightness adjusting section that adjusts brightness of at least one image signal out of the plurality of image signals corresponding to the plurality of fluorescent light beams using brightness of the image signal corresponding to the reflected light of the reference light as a reference based on a detection result by the brightness detection section.
