A fibrinolytic enzyme was produced by Pseudomonas putida B-18, isolated from soil. Screening of protease enzyme activity checked on milk nutrient agar, followed by fibrin agar plate for its fibrinolytic activity. Soy meal wheat powder broth used for the production of enzyme. Enzyme purification was done by ammonium salt precipitation, dialysis and DEAE cellulose. Native PAGE performed for the determination of molecular weight of the enzyme. In vitro blood clot lysis found within 19h of incubation. Effect of various environmental factors on enzyme activity was performed; in that maximum activity observed at 450 C temperature, pH 9.0. FeSO4, MgSO4, ZnSO4, CoCl2 and CuSO4 had inhibitory action except CaCl2.