Today, thrombosis is one of the most widely occurring diseases in modern life. Drugs with thrombolytic functions are the most effective methods in the treatment of thrombosis. This study investigates isolation of fibrinolytic enzyme producing Aspergillus sp and screening by fibrin plate. The production of extracellular fibrinolytic enzyme synthesis were carried out by using Aspergillus sp S2 were evaluated under different fermentation parameters by employing submerged fermentation method. The fibrinolytic enzyme producers were detected by the enzyme hydrolytic zone around the colony by fibrin plate assay method. Aspergillus sp S2 is the potential strain among the fungal isolates. The yield of fibrinolytic enzyme synthesis were increased after the optimization of fermentation parameters. The optimum pH 6.0, temperature 400C and inoculum size 1.25 ml and it showed 180 U/ml.