Streptokinase is a novel bacterial enzyme that binds with plasminogen and activates it. Streptokinase is produced by different species of streptococcus. Activity of streptokinase is determined by different methods such as fibrin clot method, casein hydrolysis and Chromozym test. High production of streptokinase was obtained in experiments with batch cultures. Production of streptokinase increases 2-3 times in continuous culture. Different species of streptococcus was selected for Optimum production of streptokinase by using different concentration of substrates such as CSL, molasses, sugarcane bagass, rice polishing. Maximum activity was overserved by 0.3% CSL, 0.5% molasses, 0.4% sugarcane bagass and 0.5% rice polishing. Physical parameters such temperature, pH at optimum conditions (370C, 7.0) enhance the streptokinase activity by using RSM. Nitrogen sources, carbon sources, incubation time, Growth factors such as glycine, thiamine and traces elements such as FeSO4, MgSO4, when uses in limited amount enhance the enzyme production. 1% (v/v) inoculum increase the yield of streptokinase (0.360 U/ml).