Abstract: OBJECTIVE To establish a method to evaluate the anticoagulant activity of earthworms and corresponding preparations based on the measurement of fibrinogen-thrombin time with automatic hemagglutination analyzer. METHODS Pheretima guillelmi extracts was used as test sample, and BCA method was applied to measure the content of soluble protein, and fibrinogen-thrombin time method was established to evaluate the antithrombin activity of earthworm, using the ratio of active concentration to soluble protein concentration as the index. RESULTS The protein content of ten bathes earthworms was (26.56±3.57)%. The specific activity of these earthworms was (34.64±3.98)U·μg^-1, which was similar to the activity of lumbrokinase (43.60 U·μg^-1), and 1.5-fold of purchased earthworm enzyme (22.17 U·μg^-1). CONCLUSION BCA method is applied to measure the soluble protein content of earthworms, and it is verified to be reliable and convenient. The established fibrinogen-thrombin time method express to be convenient operation, objective result and real-time measurement properties, with satisfactory methodological evaluation results, and can be used to evaluate the bioactivity of earthworms and related preparations.