The invention discloses a monomolecular fluorescence resonance energy transfer method based on photo-activation, and belongs to the technical field of biological macromolecule imaging. A photo-activated fluorescent dye is used to label a biological sample taken as the donor. An activation light source with a wavelength of 200 to 450 nanometers is used to trigger the photo-activated fluorescent dye to carry out chemical reactions such as cis-trans isomerism reactions, electron transfer reactions, chemical bond breaking reactions, and the like. The photo-activated fluorescent dye is converted from a non-activated state that does not emit fluorescence into an activated state that can emit fluorescence. Then an excitation light source with a wavelength of 450 to 1200 nanometers is used to excite the fluorescent dye so as to emit fluorescence. When the distance between a donor and an acceptor labeled by a non-photo-activated fluorescence dye is in a range of 1-10 nanometers, the energy of the donor is transferred to the acceptor, namely fluorescence resonance energy transfer. The method has the advantages that in a conventional fluorescence resonance energy transfer technology, the concentration of a fluorescence sample should reach about 50 nM, while in the provided method the measurement of monomolecular fluorescence resonance energy transfer can be carried out under a micro-mole concentration, which is close to the physiological conditions.