Abstract: In order to further increase the production of L-tryptophan fermented by Escherichia coli,response surface methodology design were used to optimize the composition of original fermentation medium. Based on the conditions,the feeding mode of ammonium acetate-corn syrup was established. The results showed that the combination of optimized medium were as follows:0.12% ammonium sulfate,0.7% potassium dihydrogen phosphate,0.15% citric acid monohydrate,0.28% magnesium sulfate heptahydrate,0.05% yeast powder 0810,0.39% ammonium acetate,0.3% corn syrup. The validation culture in 5 L tank showed that corn syrup and ammonium acetate were the main factors affecting the production of L-tryptophan. After optimization,the yield of L-tryptophan increased by 35.4%,reaching 24.53 g/L at the end of fermentation. The yield of L-tryptophan in per unit cell increased by 19.8%,reaching 0.272 g/OD. The conversion rate of glucose was increased by 27%.And it would provide some reference for L-tryptophan fermentation.