Abstract: Objective:To increase the yield of polymalic acid,reduce the process and reduce the cost. Methods:The optimal seed and culture medium were optimized by orthogonal experiment. The addition time and amount of Triton were determined by shaking flask experiment. Single factor experiments of rotational speed,ventilation volume and pH were carried out in 5 L fermentation. The best feeding medium was determined by cutting experiment in 5 L fermentor. Poured out 2 L fermented liquid when the glucose concentration was less than 20 g/L,and added 2 L different concentrations culture medium which was killed bacteria into the fermentation. Results:The optimal fermentation conditions on 5 L fermentation were confirmed:500 r/min,ventilation 5.5 L/min,pH controlled at 4.5. Added 0.4 mmol/L Triton to improve cell permeability,when fermentation lasted for 24 h,the total amount of Polymalic acid obtained in one batch reached 318 g. After processing the fermentation,pullulan was removed by 10% methanol. Then the Polymalic acid precipitation was finally obtained by adding 4 volumes of methanol precipitation. The yield was 80.52%. Conclusion:The yield of polymalic acid per liter of fermentation broth increased by 46.8%. The fermentation time of single batch increased by 40 h,and the total amount of polymalic acid was 2.25 times as much as that of the original batch. The fermentation cost was reduced,the process of the extraction was reduced in this study. So this study laid a sourd foundation for industrialited production.