The heparin affinity chromatography is widely applied for purification of proteins due to its high specificity and ease of operation. The heparin affinity chromatography medium was prepared through Schiff base method using heparin as the ligand, which was based on the macroporous polyacrylate microspheres. Firstly, the epoxy groups in the macroporous microspheres were hydrolyzed to be o-hydroxy through 0.5 mol/L H2SO4 aqueous solution. Secondly, the o-hydroxy was further oxidized to be aldehyde groups. Finally, the heparin was immobilized in the macroporous microsphere through the reaction between aldehyde groups and amino groups. The effects of operating conditions on the coupling reaction were investigated and optimized including the concentration of heparin, pH, buffer concentration, and reaction time. The effect of the reaction factor on the adsorption of proteins was evaluated using lysozyme as model protein. The optimal reaction conditions were found and the static binding capacity of the model proteins reached 40.3 mg/mL. This value was about 36% higher than that of the commercial GP-heparin. The protein recovery in this medium reached 95% with 1.0 mol/L NaCl as elution solvent. The morphology of the microspheres was observed by scanning electron microscopy. The result showed that the throughput pores were maintained in the affinity medium. The dynamic binding capacity of lysozyme on the affinity support was determined under different flow rates (31.8~318 cm/h). The result indicated the capacity at 318 cm/h decreased by about 12% in comparison with that at 31.8 cm/h. The dynamic binding capacity remained 81% of the initial value after 10 cycles. The synthesized affinity medium was used to isolate lactoferrin from mixtures. The results showed that it had a high separation efficiency.