The invention provides a preparation method of surface-modified fluorescent magnetic polymer composite microspheres. The method comprises the steps of preparing a dextran-modified nano-scale Fe3O4 dispersion liquid, preparing a mixed surfactant, preparing an oil phase, preparing an emulsion, preparing an aqueous phase, preparing a magnetic nano-scale Fe3O4 copolymer composite micro-gel, preparing SiO2-coated nano-scale Fe3O4 copolymerized magnetic polymer composite microspheres, and preparing the surface-modified fluorescent magnetic polymer composite microspheres. The prepared surface-modified fluorescent magnetic polymer composite microspheres are tested by using a fluorescence microscope, a scanning electron microscope, a fluorescence spectrometer, and a vibrating sample magnetometer; the test results show that the microspheres have the characteristics of high fluorescent luminous emissivity, good magnetic responsibility and stable performance, the microspheres have the rigidity and stability of inorganic materials and the flexibility of organic materials, and the microspheres can be applied to the aspects of immunoassay, drug carriers, immobilized enzyme, single nucleotide polymorphism genotype, cytokine identification, single-cell analysis and the like.