Antithrombin III (AT Ⅲ) is the most important anti-clotting substance. Recombinant human antithrombin Ⅲ (rhAT Ⅲ) expressed in transgenic goat milk attracts more and more attention. Develop an effective purification route for rhAT ⅢI is vital to its industrial production. An efficient purification method was developed for the rapid purification of rhAT Ⅲ by isoelectric precipitation and heparin affinity chromatography.?First, casein was effectively removed by isoelectric precipitation. rhAT Ⅲ was further purified by heparin affinity chromatography.?In the process of heparin affinity chromatography, the effects of pH and temperature on the stability of rhAT Ⅲ were studied, and the effects of operating conditions, elution gradient, flow rate and sample loaded, on the purification efficiency were also studied.?Under the optimized conditions, the protein recovery of rhAT Ⅲ was about 90% with purity over 99%, while its activity recovery was about 50%. Such a purification process?is?very simple and effective, and it would provide a valuable reference for the further scaling-up of industrial production.