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 29.3-29.5 kD 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.