To optimize the preparation process of fibrinolytic enzyme(AprE9912D) lyophilized powder，and to explore its fibrinolytic mechanism.Methods:The purified AprE9912D was processed by vacuum freeze-drying with different proportions of excipients.The optimal lyophilized powder formula was selected by enzyme activity，appearance，and redissolution time.The optimal storage temperature of lyophilized powder was determined by an accelerated stability test.The enzyme activity of AprE9912D was detected in vitro in standard fibrin plates and heated fibrin plates.Molecular docking was used to predict the interaction between AprE9912D and fibrinolytic target proteins.Degradation of fibrin，fibrinogen，and gelatin by AprE9912D was also tested in vitro.Results:The best compound formula of AprE9912D lyophilized powder was 1% glycine and 3% sucrose，with a white and loose cake shape.Stored at -20 ℃ for 150 days，the appearance qualification rate was 92.9%，the enzyme activity loss rate was 8.4%，and the redissolution time was 3.0 s.The optimal storage temperature for AprE9912D lyophilized powder was less than or equal to -20 ℃.The molecular docking experiment found that the affinity sequence of AprE9912D against thrombus target proteins was fibrinogen>fibrin>plasminogen.By in vitro fibrin plate and degradation experiment，it was verified that AprE9912D had direct fibrinolytic activity and an indirect plasminogen activation effect，with a higher affinity for fibrin.The fibrinolytic mechanism was also possibly related to anti-collagen-induced platelet aggregation.Conclusion:The formula and preparation process of AprE9912D lyophilized powder are feasible.The fibrinolytic mechanism is preliminarily analyzed in vitro，laying the foundation for further development into fibrinolytic medicines.