A fluorescence observation apparatus comprising: a light source section that can emit a plurality of excitation light beams for exciting a plurality of fluorescent substances and a reference light having a narrow wavelength band; 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 (18, 19) that generates image signals 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, sets an upper limit value and a lower limit value of brightness using brightness of the image signal corresponding to the reflected light of the reference light as a reference and detects whether or not the brightness of the plurality of image signals corresponding to the plurality of fluorescent light beams falls within a range between the upper limit value and the lower limit value; a brightness adjusting section that adjusts brightness of each image signal outside the range out of the plurality of image signals corresponding to the plurality of fluorescent light beams so as to fall within the range, while adjusting a color tone, based on a detection result by the brightness detection section; and an image synthesizing section that assigns each of the image signals subjected to color tone adjustment by the brightness adjusting section to a different color channel and synthesizes images. a light source section that can emit a plurality of excitation light beams for exciting a plurality of fluorescent substances and a reference light having a narrow wavelength band; 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 (18, 19) that generates image signals 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, sets an upper limit value and a lower limit value of brightness using brightness of the image signal corresponding to the reflected light of the reference light as a reference and detects whether or not the brightness of the plurality of image signals corresponding to the plurality of fluorescent light beams falls within a range between the upper limit value and the lower limit value; a brightness adjusting section that adjusts brightness of each image signal outside the range out of the plurality of image signals corresponding to the plurality of fluorescent light beams so as to fall within the range, while adjusting a color tone, based on a detection result by the brightness detection section; and an image synthesizing section that assigns each of the image signals subjected to color tone adjustment by the brightness adjusting section to a different color channel and synthesizes images.