A Bacillus amyloliquefaciens strain GSBa-1 was isolated from sweet Jiuqu. In order to improve the ability of producing a milk-clotting enzyme (MCE) by liquid fermentation of strain GSBa-1, the single factor test and response surface methodology were employed to optimize the composition of the medium used. The single factor experiment was carried out to analyze the influence of different carbon source, nitrogen source, metal salt and phosphorus source on production of MCE by strain GSBa-1. Then the Box-Behnken design based RSM experiment was used to study the optimum combination of three main factors such as the content of maltose, peptone and yeast extract powder in the medium. The optimal medium composition for producing MCE by GSBa-1 was as follows:maltose 1.93g/L, peptone 10.89g/L, and yeast extract powder 2.15g/L. When the optimal culture medium was used, MCE activity was up to (562.57±7.67)Su/mL, which is similar as the theoretical prediction value of 537.10 Su/mL with an average error of 4.53%. After optimization of the medium composition, the MCE activity produced by strain GSBa-1 increased by 1.88 times compared with that of the basal medium.