With Threonine deaminase (L-TD) and leucine dehydrogenase (L-LeuDH) obtained by cloning and expression as biocatalyst, the natural amino acid was converted into unnatural amino acid using the "one-pot" process. Finally, the product was obtained by crystallization with a total yield of 84%.The configuration of the product obtained was confirmed by LC-MS and 1H-NMR, and the ee > 99.9%. This process uses cheap raw materials and enzymes, and avoids the intercurrent extraction and separation, providing a good prospect for an efficient and low-cost 2-aminobutyric acid production process.