Abstract: To optimize the fermentation medium of CMP-sialic acid synthase(NmCSS)fermented by Streptococcus agalactiae with low cost and high enzyme activity,the ferment temperature,carbon source,nitrogen source and pH were selected by single factor experiment to determine the main factors and level values which had significant influence on the enzyme activity of NmCSS. Box-Behnken response surface design was used to determine the optimal concentration for nitrogen source,carbon source and pH of medium. The results showed that the enzyme activity of NmCSS could be up to(5.895±0.005)×10⁷ U/mol when the temperature was 34 ℃,sucrose concentration was 0.10%,nitrogen source concentration was 2.50%,pH values was 7.5,Na₂HPO₄ was 2.5 g/L and NaCl was 5.0 g/L. After fermentation verification,the enzyme activity of NmCSS was 10.627 times higher than the enzyme activity of(0.507±0.002)×10⁷ U/mol under basic fermentation medium conditions. The low cost and high enzyme activity medium were optimized by response surface method,which laid a foundation for the production and application of NmCSS from Streptococcus agalactiae.