Abstract: The optimal flask-shaking batch fermentation medium of microbial transglutaminase produced by Streptomyces sp. HS-1 was studied by Plackett-Burman design and Response Surface Analysis (RSA) with SAS (Statistical Analysis System). The results showed that the effects of soybean hydrolysate, glycerol and ammonium sulfate on the activity of microbial transglutaminase were significant among the eight factors by Plackett-Burman design and analysis. A polynomial regression model including the above three important factors was set up by RSA. The optimal concentrations of the three factors were assessed by the Canonical analysis. Estimated optimal conditions of the three factors were as the follows: soybean hydrolysate 24.0 g #8226;L-1, glycerol 24.4 g #8226; L-1 and ammonium sulfate 5.40 g #8226; L-1. The enzyme activity was increased by 16.4%, from 6.27 μ #8226; mL-1 to 7.30 μ #8226; mL-1 using the optimal flask-shaking batch fermentation medium.