Abstract: Chitosan beads were prepared by inverse suspension crosslinked polymerization.Chitosan beads, activated by epichlorohydrin, that were coupled with heparin to prepare affinity chromatography reagent.The prepared affinity chromatography reagent of heparin-chitosan beads was used to purify porcine thrombin.Results showed that optimum parameters of preparing chitosan beads were 112.5mL of 5% chitosan solution, 75mL of liquid paraffin, 15mL of acetic ether, 10mL of formaldehyde, 2mL of glutaraldehyde, 8 drops of Span 80 and stirring speed of 2500r/min.After swelling with ddH 2 O for 24h, the prepared chitosan beads showed apparent porosity.Detection results of porcine thrombin activity confirmed that the affinity chromatography of heparin-chitosan beads could be used in the purification of porcine thrombin.These results could present some references for the preparation of affinity chromatography reagent and the purification of porcine thrombin.