Abstract: In order to exploit fibrinolysis components of Chinese traditional bacteria-type Douchi, separation and purification of fibrinolytic enzyme were discussed and the best purification method was determined. Firstly, fibrinolytic enzyme was separated by ammonium sulfate with concentration of 75% saturation. Secondly, purification was achieved by Diethylaminoethyl Sepharose Fast Flow(DEAE-Sepharose FF) anion exchange chromatography with 10mmol/L Tris-HCl (pH 8.7) as sample buffer. The column was eluted stepwise with 0.6 mol/L NaCl and 10 mmol/L Trishydroxymethylaminomen(Tris-HCl) buffer (pH 8.7) at flow rate of 1 mL/min. Finally, fibrinolytic enzyme was further filtered by Sephadex G-75 chromatography and 11.29 times purified fibrinolytic enzyme was gained. Sodium dodecyl sulphate-polylamide gel electroresis (SDS-PAGE) showed that there was no unpurposed protein in purified fibrinolytic enzyme and the molecular weight was identified approximately 31ku preliminarily.