A fibrinolytic enzyme from Agrocybe aegerita and a preparation method thereof. The fibrinolytic enzyme from Agrocybe aegerita is composed of two subunits A and B, the molecular weight of the subunit A is 31.4 kDa; the molecular weight of the subunit B is 21.2 kDa; the twelve amino acids sequence of the N-terminal of the subunit A is shown in SEQ ID No. 1; the twelve amino acids sequence of the N-terminal of the subunit B is shown in SEQ ID No. 2. The optimal temperature for the fibrinolytic enzyme from Agrocybe aegerita provided in the invention is 47 C., which can maintain good activity at physiological pH of human. Fe.sup.2+ at different concentrations shows obvious inhibiting effects on the fibrinolytic enzyme from Agrocybe aegerita. The fibrinolytic enzyme from Agrocybe aegerita exhibits good thrombolytic properties, thus providing a research basis for the preparation of thrombolytic drugs and/or functional foods.