Abstract: To obtain a strain with high Alkaline phosphatase activity from the soil,and to optimize the best fermentation conditions. A strain with high potential for secreting Alkaline phosphatase was isolated through Alkaline phosphatase reaction plate and fermentation screening. Based on strain morphology,physiological and biochemical characteristic and molecular biology assay,the strain has been identified as Acetobacter oriental S377. According to single factor experiment,taking the fructose,urea,calcium carbonate and pH as the experimental factors,the response surface test was optimized by the center combination experiment with the Alkaline phosphatase activity as the response value. The result revealed that the optimal fermentation conditions was fructose 8.9 g/L,urea 0.4 g/L,calcium carbonate 4.5 g/L and the initial pH9.2.The activity of Alkaline phosphatase of S377 could reach to(28600±65) U under the optimization conditions,which was approximately 6.4 times as that at the original culture conditions. The enzyme activity of S377 was significantly higher than that of other microorganisms reported,which provided a new microbial resource for the production of Alkaline phosphatase and was of great significance to the industrial production of this enzyme.