Abstract: Two thrombin-like enzymes (principle Ⅰ and principle Ⅱ) had been purified from the venom of Trimeresurus stejnegeri by CM-Sephadex C-25,DEAE-Sephadex A-50 and DEAE-Sepharose CL-6B column chromatography.Principle Ⅰ showed one band and principle Ⅱ two bands on polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis.We had identified the two bands of principle Ⅱ both were thrombin-like enzymes by the method of cutting polyacrylamide gel electrophoresis.Principle Ⅰ has a molecular weight of 54,500,contains approximately 261 amino acid residues and has a relatively high content of aspartic acid and glutamic acid,contains 14% neutral hexose,13.1% hexosamine and 14.7% sialic acid,the isoelectric point is 3.5,the extinction coefficient at 280 nm for a 0.1% solution is 0.855.Principle Ⅱ has molecular weights of 54,000 and 47,000 respectively as determined by SDS-polyacrylamide gel electrophoresis,principle Ⅱ is also a glycoprotein as showed by Schiffs stain.