Background Hemocoagulase Agkistrodon for injection is a single component thrombin which has passed phases I and II clinical trials. The purpose of this phase III clinical trial was to evaluate the effect of Hemocoagulase Agkistrodon on hemostasis and coagulation in abdominal skin and subcutaneous incisions and to assess the safety of this agent in surgical patients. Methods This is a phase III, prospective, randomized, double-blind, and controlled multicenter clinical trial including 432 consecutive patients randomized into either a study group (injected with hemocoagulase Agkistrodon at 2 U, n=324) or a control group (injected with hemocoagulase Atrox, n=108). The hemostatic time, hemorrhagic volume, hemorrhagic volume per unit area, blood coagulation, and adverse events were measured and compared between the two groups. Results The mean hemostatic time in the study group was (36.8±18.7) seconds; the hemorrhagic volume was (3.77±3.93) g; and the hemorrhagic volume per unit area was (0.091±0.125) g/cm~2. In the control group, the corresponding values were (38.1±19.7) seconds, (4.00±4.75) g, and (0.095±0.101) g/cm~2, respectively. No significant difference in values existed between the two groups (P >0.05). Blood coagulation results and hepatic and renal function were also similar between the two groups. Adverse events were reported in two cases, but were deemed non-drug-related. Conclusions Hemocoagulase Agkistrodon has good hemostatic and coagulative function and is safe for the use of arresting capillary hemorrhage that occurs while incising the abdomen during surgery.