D-alanine is an important pharmaceutical intermediate. In this study, an effective method was developed to prepare optically pure D-alanine from sodium pyruvate with co-immobilized meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum and formate dehydrogenase from Candida Boidinii onto a resin. After optimizing the conditions of immobilization and biotransformation, the co-immobilized enzymes exhibited excellent stereoselectivity, yield and reusability. The co-immobilized enzymes kept 80% of its activity after 12 reuses, and o-alanine was isolated in 75.7% average yield and 99.5% ee for the first 20 cycles in the batch reactions. This suggests the co-immobilized enzyme system as a promising biocatalyst for the green production of o-alanine from pyruvic acid, a bio-based starting material.