A fluorescence observation unit includes a scanner that scans ultrashort pulsed laser light, a pupil projection lens that focuses the scanned ultrashort pulsed laser light, an image-forming lens that converts the focused ultrashort pulsed laser light to substantially collimated light and causes the ultrashort pulsed laser light to be incident on the objective lens, and a dichroic mirror that splits off, from the optic path of the ultrashort pulsed laser light, fluorescence generated in a sample due to irradiation with the ultrashort pulsed laser light and collected by the objective lens. The image-forming lens includes a first optical system having positive refractive power, and a second optical system having negative refractive power and disposed at a position closer to the scanner than the first optical system is. The dichroic mirror is disposed between the first optical system and the second optical system.