Intravascular thrombosis is a prime cause of cardiac complications worldwide. Microbial fibrinolytic proteases are of clinical significance in thrombosis treatment. The present study discusses the purification and characterization of a protease from Bacillus cereus S46, ascertaining its in vitro thrombolytic activity against a blood clot. By the three-step purification involving precipitation, dialysis, and diethylaminoethyl-cellulose ion-exchange chromatography, a 12.37-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the protease was 30 kDa, as found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme was observed at pH 8.0 and 40°C. The enzyme retained an 82.19% residual activity at pH 8.0 and 40°C for 1 h. The K and V values of the protease with casein were 0.0027 mM and 9.712 µmol/min, respectively. In an in vitro assay, the purified protease resulted in 97.02% lysis of the blood clot. The fibrinolytic potential of the enzyme, together with its characteristics of being active and stable under near-physiological conditions, may suggest its application as a therapeutic agent.© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.