D-Alanine (D-Ala) is an important chiral amino acid with a wide range of applications in the fields of medicine, food, and chemical industry. In this project, a "two-bacteria, four-enzyme" catalytic process for the preparation of D-Ala, a fed-batch fermentation process with pRMA [E.coli BL21(DE3)Alr-Dadx--pRSFDuet-1-MaiA-AspA] and pEAD2 [E.coli BL21(DE3)Alr-Dadx--pETDuet-1-AspR-DaaT] trains was established to achieve co-expression of maleic acid cis?trans isomerase (MaiA)/asparaginase (AspA) and aspartate racemase (AspR)/D-amino acid transcarbamylase (DaaT). Additionally, process parameters for multiple enzyme preparation of D-Ala with maleic anhydride (MA) as the substrate were optimized, including temperature, pH, and concentrations of pyruvate, pyridoxal phosphate, maleic anhydride, and pRMA/pEAD2 cells, leading to the development of an efficient enzymatic conversion process for D-Ala. The results showed that the cell concentration and apparent activity of pRMA reached the maximum value of 72.56 g/L and 554.49±30.96 U, respectively, at 23 h. In contrast, the apparent activity of pEAD2 could reach 513.74±38.25 U at 9 h, and the cell concentration was 30.75 g/L. For the multi-enzyme preparation of D-Ala, the optimized catalytic system was composed of 1.5 mol/L MA, 5.0 mmol/L pyridoxal phosphate and 3.52 g/L pyruvic acid. The dosages of pRMA and pEAD2 cells were 6.60 g/L and 9.80 g/L, added at 3 h intervals. The optimum reaction conditions were pH=8.0, temperature of 50℃, and rotation speed of 200 r/min for 24 h. The substrate conversion rate was up to 99.00%, and the yield of D-Ala reached 93.97%. Under the above optimal conditions, when the reaction volume was scaled up 40-fold, from 100 mL to 4 L, there was no significant difference in substrate conversion and product yield, which laid the foundation for its industrial application.