L-2-Aminobutyric acid can be synthesized in a transamination reaction from L-threonine and L-aspartic acid as substrates by the action of threonine deaminase and aromatic aminotransferase, but the by-product L-alanine was produced simultaneously. A small amount of L-alanine increased the complexity of the L-2-aminobutyric acid recovery process because of their extreme similarity in physical and chemical properties. Acetolactate synthase has been introduced to remove the pyruvate intermediate for reducing the L-alanine concentration partially. To eliminate the remnant L-alanine, alanine racemase of Bacillus subtilis in combination with D-amino acid oxidase of Rhodotorula gracilis or Trigonopsis variabilis respectively was introduced into the reaction system for the L-2-aminobutyric acid synthesis. L-Alanine could be completely removed by the action of alanine racemase of B. subtilis and D-amino acid oxidase of R. gracilis; thereby, high-purity L-2-aminobutyric acid was achieved. The results revealed that alanine racemase could discriminate effectively between L-alanine and L-2-aminobutyric acid, and selectively catalyzed L-alanine to D-alanine reversibly. D-Amino acid oxidase then catalyzed D-alanine to pyruvate stereoselectively. Furthermore, this method was also successfully used to remove the by-product L-alanine in the production of other neutral amino acids such as L-tertiary leucine and L-valine, suggesting that multienzymatic whole-cell catalysis can be employed to provide high purity products.