To efficiently detect fluorescence emitted from a sample, and to detect a plurality of wavelengths of fluorescences concurrently by concurrent irradiation with a plurality of wavelengths of excitation lights.

This laser scanning fluorescence observation device 1 is provided with a laser light source 2 having laser beams of the plurality of wavelengths, a spectrograph 5 for dispersing spectrally the laser beams into every of the wavelengths, a convergence lens 6 for converging the spectrally dispersed laser beam, a wavelength selective reflection device 7 provided integrally with a plurality of reflection parts arranged with a prescribed space along a spectral dispersion direction in the vicinity of a focal point position thereof to reflect respectively the laser beams different in the wavelengths, and a transmission part arranged adjacently thereto, a diffraction grating 13 for associating the reflected laser beams, a scanning part 24 for scanning two-dimensionally the associated laser beam, an objective optical system 21 for converging the scanned laser beam in a sample A, and a photodetector 12 for detecting the fluorescences returned from the sample A. A width dimension along a spectral direction is thinner than that of the transmission part, in each of the reflection parts of the wavelength selective reflection device 7.