To provide a fluorescence detection device that can effectively remove autofluorescence using a simple configuration. A fluorescence detection device comprises: semiconductor laser for emitting excitation light; an objective lens for converging excitation light onto a sample on a biosensor substrate; an anamorphic lens which introduces astigmatism to fluorescence that is from the biosensor substrate and incident on the objective lens and that passed through the objective lens; a spectral element for separating the fluorescence into a plurality of light rays; and a fluorescence detector for receiving the light rays separated by the spectral element. On the light receiving surface of the fluorescence detector, the spectral element splits the fluorescence so that the fluorescence generated at the sample is separated from the fluorescence generated at a specific depth position other than the sample position.