Streptokinase (SK) is a fibrinolytic protein which has been produced by several species of β-hemolytic Streptococci. It has been extensively used to treat cardiovascular disorders by activating plasminogen into plasmin and eventually dissolving the blood clot. The objective of the current research project was to exploit indigenously isolated Streptococcus agalactiae EBL-20 for the production of SK and enhancing its productivity by Ultraviolet (UV) irradiation and ethyl methanesulfonate (EMS) induced mutations, optimized culture conditions and nutritional requirements. Four mutants namely, S. agalactiae EBL-31, S. agalactiae EBL-32, S. agalactiae EBL-33 and S. agalactiae EBL-34 were selected by different screening procedures using triton X-100, casein plasminogen overlay assay and clot lysis assay. The optimum levels of four process variables (pH, temperature, incubation time and inoculum size) were obtained by Response Surface Methodology (RSM). According to statistical results, a temperature value of 37.5⁰C and pH 7 of the fermentation medium with 2.50 mL inoculum size for 36 hours of incubation was considered as optimum for enhanced SK production. After optimization, mutant derived strains EBL-31, EBL-32, EBL-33 and EBL-34 resulted in 2.01, 2.30, 3.70 and 2.48 fold increased activity as compared to parent S. agalactiae EBL-20, respectively. The best mutant obtained after performing optimization studies was found to be S. agalactiae EBL-33 with 3.70 fold increased activity, hence it was purified and exploited for characterization studies with reference to S. agalactiae EBL-20.The purification of SK by DEAE-cellulose and Sephadex-G100 resulted into specific activity of 447.6 and 1278.87 U mg⁻¹ from parent S. agalactiae EBL-20 and mutant derived S. agalactiae EBL-33, respectively. The molecular weight of SK obtained from both the strains of S. agalactiae was found to be 47 kDa. Study of kinetic and thermodynamic parameters revealed that SK obtained from S. agalactiae EBL-33 has more potential for utilizing its substrate i.e., plasminogen with high thermostability, in contrast to SK derived from S. agalactiae EBL-20. The study concludes that both UV rays and EMS are effective mutagenic agents for enhanced production of SK from S. agalactiae. Moreover, enhanced SK yield can be obtained by optimizing the nutritional necessities and fermentation/cultivation conditions of the microorganism.