The present invention discloses a fluorescent microsphere and its spray drying preparation method and application. The invention involves encapsulating near-infrared fluorescent substances and magnetic nanoparticles into polymer microspheres through the spray drying method, or encapsulating gold nanoparticles with magnetic nanoparticles into polymer microspheres, where the spheroidization and encapsulation are completed in one step. The microspheres can further contain anticancer drugs, and their surfaces will also be linked with biomolecules with recognition functions. The microspheres obtained by the invention can destroy tumor cells through the thermal effect generated by the near-infrared fluorescent substances or gold nanoparticles under excitation light, or simultaneously use the cytotoxic effects of anticancer drugs and the thermal effects produced by the near-infrared fluorescent substances or gold nanoparticles to treat tumors. The magnetic nanoparticles in the microspheres can locate or fix the microspheres at the tumor site. The microspheres obtained by the invention can also be used for biomedical labeling and tracing.