Subtilisin QK is a nattokinase-like serine protease which demonstrates high efficiency for direct cross-linked fibrin degradation, without activation of plasminogen. It was resistant capacity against the digestion of mimics pancreatic enzyme and could be absorbed throughout ex vivo intestine with fibrinolytic activity in a time-dependent manner. The result of bioluminescence image using nude mice also proved that rSubtilisin QK could infiltrate into all organs from intestinal mucosa. Since rSubtilisin QK could also elevated the levels of plasma levels of fibrin/fibrinogen degradation products and D-dimer (they are sensitive indicators of in vivo fibrinolytic activity) in rats, its prevention effect was investigated in κ-carrageenan-induced rat model. Thrombosis rats were oral treated with rSubtilisin QK for 7 days, and the length of tail-thrombosis was then measured. Compared to control group, the average length of tail-thrombosiswere reduced and the vessel cross section of ears and tails occupied by thrombosis was relieved among the thrombosis rats treated with rSubtilisin QK. Furthermore, the levels of tissue plasminogen activator were increased in cultured HUVECs stimulated with rSubtilisin QK, while plasminogen activator inhibitor-1 showed the opposite trend simultaneously, suggesting the activation of plasminogen. In conclusion, rSubtilisin QK exerted thrombolytic effects in vivo and may be a potential oral thrombolytic agent.