Disclosed herein is a fluorescence detection method. The fluorescence detection method includes the steps of: irradiating a measurement object with laser light modulated at a predetermined frequency; receiving fluorescence emitted by the measurement object and outputting two or more pulsed fluorescent signals; setting reference timing in units of period corresponding to the frequency; acquiring a generation time to output of each of the pulsed fluorescent signals based on the reference timing; generating a cumulative fluorescent signal indicating the relationship between a generation frequency of the pulsed fluorescent signal and the generation time; determining, by using a signal corresponding to modulation of the laser light as a reference signal, a phase difference between the reference signal and the cumulative fluorescent signal; and determining, by using the phase difference, a fluorescence relaxation time of the fluorescence emitted by the measurement object.