To study the characterization of a protease ARSP1 (apoptosis-related serine protease) of Eisenia fetida, a recombinant ARSP1 was constructed. ARSP1 was produced in E. coli BL21-CodonPlus (DE3)-RIL after IPTG induction and exited in inclusion body. After refolding in vitro, the protein was purified by DEAE-Sepharose F.F. and Sephacryl S-100 chromatography in sequence. ARSP1 showed high sequence identity to other chymotrypsin-like serine proteases and the catalytic triad was His41-Asp90-Ser188. ARSP1 could degrade casein following Michaelis-Menten kinetics with a Vmax of 43.9 U/mg protein and a Km for casein of 0.83 g/l. Studies with inhibitors indicated that ARSP1 was a chymotrypsin-like serine protease. Experiments in vitro demonstrated that ARSP1 could not only hydrolyze fibrinogen and fibrin directly, but also activate plasminogen to plasmin. ARSP1 inhibited thrombin activity and ADP-induced platelet aggregation in a dose-response correlation. These results showed that ARSP1 has thrombolytic activity and also an anti-thrombus function.