The invention relates to a method for preparing a polymer fluorescent microsphere. The method comprises the following steps of: 1, preparing polymer fluorescent microsphere liquid drops, namely dissolving a first polymer into a solvent, adding a fluorescent material into the solvent, magnetically and uniformly stirring the fluorescent material and the first polymer in the solvent to prepare a non-continuous phase, respectively putting the non-continuous phase and a continuous phase into injectors connected with a non-continuous phase inlet and a continuous phase inlet of a microfluid device, adjusting the flowing speeds of the two phases of solutions through a trace sample injection pump, so as to obtain the fluorescent microsphere liquid drops, and at a microfluid outlet, collecting the fluorescent microsphere liquid drops into a fluorescent microsphere receiving device; and 2, preparing the polymer fluorescent microsphere, namely drying the fluorescent microsphere liquid drops until the solvent is completely volatilized, then washing the fluorescent microsphere liquid drops by a washer, and cleaning the continuous phase of solution to finally obtain the polymer fluorescent microsphere. Equipment used by the method is simple and convenient to operate; the particle size of the microsphere can be adjusted by adjusting the flowing speeds of the continuous phase and the non-continuous phase; and the uniformity of the particle sizes of the prepared microspheres is high.