The main aim of this work was optimization of physical conditions as well as medium components for the maximum production of fibrinolytic enzyme. A fibrinolytic enzyme producing organism was isolated from soil and identified as Bacillus altitudinis strain S-CSR 0020. Various physical parameters such as temperature, pH, incubation time and medium components such as inoculum size, substrate (nitrogen and carbon) concentrations were optimized. A cultivation medium was designed using optimized conditions by Box Behnken Design for mass production of fibrinolytic enzyme and specific activity of enzyme was analyzed. The maximum enzyme production seen at 50 °C temperature, 10.0 pH, 4% substrate concentration with 3 ml inoculum size and 96 h. of incubation time. Among the different carbon sources tested, fructose (8%) showed maximum enzyme activity of 325 U/ml with a specific activity of 812.5 U/ml. Box Behnken Design was used to optimize the percentage of best nitrogen sources (casein, peptone, soya bean and yeast extract) with an enzyme activity of 1089.8 U/ml. Experiments were repeated with optimized condition manually and found with an enzyme activity of 1072.12 U/ml. Mass production of extracellular fibrinolytic enzyme was done with all these conditions and it showed an enzyme activity of 2070 U/ml with an initial activity of 400 U/ml without medium optimization. There was a fivefold increase in fibrinolytic activity after medium optimization that proved the reliability of optimization manually as well as using the Box Behnken design.