Abstract: A method of fluorescence imaging is disclosed that uses a non-labeled fluorescent microsphere comprising a primary amine polymer, wherein the microsphere does not contain an additional fluorescent substance, and wherein at least one primary amine group in the primary amine polymer has the structure of —N?R—, in which R is a residue after the reaction of an aldehyde-type cross-linking substance, and the other primary amine groups have the structure of —NH2. The fluorescence color of the microspheres can be adjusted by selecting different aldehyde-type cross-linking substances or combinations, and the fluorescence intensity of the microspheres can be controlled not only by the addition of the aldehyde-type cross-linking substances and the time of the cross-linking reaction, but also by adjusting the diameters of the microspheres.