A method is provided for detecting a fluorescent species that is reversibly photoswitchable at high frequency, and more precisely to a method for detecting at least one reversibly photoswitchable fluorescent species, including a step consisting in illuminating a sample containing a reversibly photoswitchable fluorescent species with a first illuminating light beam, of wavelength λ1, and periodically modulated at an angular frequency ω, and with a second illuminating light beam of wavelength λ2 different from λ1, which is periodically modulated at the angular frequency ω, the second illuminating light beam being modulated in antiphase with respect to the first illuminating light beam.