Nanometer Fe₃O₄ particle was synthesized by coprecipitation, magnetic polymeric microspheres- magnetic chitosan microspheres, containing a magnetic core and a polymer shell, were prepared by the suspension cross-linking technique. Novel affinity magnetic microspheres were obtained after immobilizing heparin onto the nanoparticles. The size, morphology, structure and magnetic response properties were studied by means of SEM, FT-IR and XRD and so on. The novel affinity magnetic microspheres′ properties for thrombin were examined by its adsorption and purification, and compared with the traditional means of column chromatography—DEAE-Sepharose Fast Flow. The results showed the affinity magnetic microspheres had a spherical appearance, narrow distribution of diameter, the particle diameter is about 50 nm, and one-step purification of thrombin using it the specific activity was 1 879.7 U/mg, recovery rate was 85% and purification fold was 11.057. Whereas, purification of thrombin by column chromatography, the product exhibits an activity of 909.84 U/mg, recovery rate of 72% and purification fold of 5.33. In conclusion, chitosan nanometer affinity magnetic microspheres was prepared and magnetic separation techniques was applied for purification of thrombin, the results is better than that from traditional methods and these results have some references values in the production and subsequent study on the research and development.