A method for the preparation of D-alanine by continuous catalyzed of aspartic acid racemase and D-amino acid transaminase was established. DL-aspartic acid was obtained by racemization of L-aspartic acid with whole cell of aspartic acid racemase, and then the whole cell was removed by centrifuging, and the residual aspartic acid racemase was inactivated by heating. Then, added D-amino acid transaminase purified by Ni-column affinity chromatography, D-aspartic acid (D-Asp) and pyruvic acid (PA) were converted to D-alanine. The optimal catalytic conditions of aspartate racemase were as follows: the reaction temperature was 40 oC, 0.2 mol/L of potassium phosphate buffer solution (pH=7.0), 100 g/L of L-aspartic acid. The optimal catalytic conditions of D-amino acid transaminase were as follows: the reaction temperature was 42 oC, 0.2 mol/L of potassium phosphate buffer (pH=7.0), 4 mmol/L of pyridoxal phosphate, 50 g/L of DL-aspartic acid, the molar ratio of PA and D-Asp is 1:10. The transformation product D-alanine was obtained by the method of isoelectric point crystallization and cationic resin separation. The conversion of D-aspartic acid was 94%, the yield of D-alanine was 84%, and the ee value of D-alanine was 98%.