THIS STUDY sought to demonstrate the optimization of streptokinase production. Enzyme production was monitored during the growth of both Streptococcus pyogenes and Streptococcus equisimilis in different media. Adjustment of the pH for culture media of S. pyogenes and S. equisimilis, every 12 hr during incubation, significantly increased the enzyme production levels, when both microbes were grown on Strep-base medium. The best carbon source for streptokinase production was glucose of both S. pyogenes and S. equisimilis, while mannitol and sorbitol were found to be less suitable carbon sources. Yeast extract and casein could be used as the primary source of organic nitrogen for streptokinase production when the microbes were allowed to grow on Strep-base medium. The highest levels of the enzyme production were obtained with 1.5 % (w/v) tryptone and 1.5 % (w/v) casein for S. equisimilis and S. pyogenes, respectively. Detection of streptokinase produced was by the common casein digestion method and by the more sensitive chromozym substrate digestion method. Moreover, the enzyme was assayed electrochemically using the protamine-sensitive electrode to compare different methods of detection. Results obtained from electrochemical method were very close to that obtained with other methods. These results offer an alternative and reliable method for streptokinase detection during microbial growth. It provides a faster and less expensive technique for streptokinase determination especially when there is a need to detect the enzyme in turbid media.