The main focus of this current research is to enhance the efficiency of Streptococcus equisimilis (MTCC3522) using random mutagenesis for enhanced streptokinase production. The influence of environmental parameters for the growth and streptokinase production of the UV mutated strain UVM6 was determined by optimizing various conditions. Five different parameters were assigned for optimization studies carbon source, salt concentration, pH, temperature and growth medium. The maximum streptokinase activity of 0.3U/ml was obtained in the culture conditions, glucose (1%) as carbon source, 2.5g/l, NaCl concentration, at 37°C for 24 h with an initial pH 7.5 at 120 rpm. The blood clot lysis by mutated strain exhibited maximum enzyme activity. The maximum production of streptokinase activity was shown by mutated strain Streptococcus equisimilis UVM6. The results showed that UV irradiation was one of the effective mutagenic agents for strain improvement studies of Streptococcus equisimilis for streptokinase production.