A thrombin-like enzyme isolated from Agkistrodon acutus venom, comprising an alpha subunit having the sequence of SEQ ID No.1 and a beta subunit having the sequence of SEQ ID No. 2, which are linked by seven disulfide bonds, is provided. The hemocoagulase of Agkistrodon acutus in the present invention is a serine proteinase having a molecular weight of 29.3-29.5kD and an isoelectric point of 5.5, and is able to hydrolyze the alpha chain of human fibrinogen. The invention also provides methods of purifying the thrombin-like enzyme from snake venom, which comprise removing insoluble substance by pretreatment, conducting twice of anion-exchange column chromatography, collecting active eluting peak, dialyzing, ultra-filtrating and desalinating so as to obtain a snake venom thrombin-like enzyme and the enzyme has a specific activity of no less than 180u/mg protein, a HPLC purity of more than 95% and a yield of 0.7%-0.8% based on the weight of the snake venom raw material.
