Different medium components were screened to improve the productivity of the novel bioactive compound, cystocin from the Streptomyces sp. GC0001. Plackett and Burman statistical design was employed to screen the effective components. Finally, response surface methodology based on three factors Box-Behnken design was applied to optimize the limiting variables such as soytone, glucose and magnesium sulfate concentration. The antibiotic yield was increased accordingly with the concentration of soytone and glucose. Magnesium sulfate has vital role in productivity besides the other carbon and nitrogen sources. Pharmamedia retained the strongest negative effect for the production of antibiotic and the effect due to sucrose and calcium carbonate was minor. The optimal concentrations of medium components for the cystocin production are determined as; soytone (50 g/L), glucose (40 g/L) and magnesium sulfate (30 g/L).