A total internal reflection fluorescence microscope includes a light source which generates laser light having a plurality of wavelengths, a focal optical system which irradiates the laser light from the light source on a specimen at a predetermined incident angle via an objective lens and which generates evanescent illumination, a fluorescence observation unit which observes fluorescence generated from the specimen due to the evanescent illumination, an incident angle adjuster which adjusts the incident angle of the laser light irradiated on the specimen, and a controller which controls the incident angle adjuster such that the amount of the permeation depth of the evanescent light is the same when the wavelength of the laser light from the laser light source is switched.