A novel fibrinolytic enzyme was isolated and purified from the fruiting bodies of Lyophyllum decastes. The N-terminal amino acid sequence of the enzyme was determined by Edman degradation method.The fruiting bodies of Lyophyllum decastes was dried, crushed and extracted with phosphate solution to obtain crude enzyme solution.The crude enzyme solution was isolated and purified by using ammonium sulfate precipitation, Octyl-Sepharose Fast Flow hydrophobic chromatography, SP-Sepharose high performance ion chromatography and Source 15PHE hydrophobic chromatography to obtain a single component with fibrinolytic activity.The specific activity of the enzyme was 4 105.78 U/mg, the purification fold was 206.09, and the recovery rate of the activity was 30.91%.Native-PAGE and SDS-PAGE showed that the fibrinolytic enzyme reached electrophoretic purity and the molecular weight was 30.9 kDa.The N-terminal sequences of the fibrinolytic enzyme were determined by the Edman degradation method, and the sequences was Gly-Ala-Val-Thr-Gln-Cys-Asn-Ala-Pro-Trp-Gly-Leu.By NCBI database comparison, it was found that the fibrinolytic enzyme was a novel fibrinolytic enzyme.The research provides a new idea and method for the research and development of fibrinolytic enzyme.