L-2-aminobutyric acid (L-ABA) is an important pharmaceutical intermediate and food additive. In order to achieve the efficient biosynthesis of L-ABA, two recombinant Escherichia coli strains carrying threonine deaminase, leucine dehydrogenase and different formate dehydrogenases encoding genes were established by using a dual plasmid co-expression system of pACYCDuet-1 and pET28a, which were named as E. coli BL21(DE3)/ pACYCDuet-1-EsLeuDH-EcTD: pET28a- CbFDH and E. coli BL21(DE3)/ pACYCDuet-1-EsLeuDH-EcTD: pET28a- CbFDHM, respectively. After induction, the two recombinant E. coli strains successfully expressed three target enzymes. The results showed that the expression levels of L-threonine deaminase and L-leucine dehydrogenase in the two recombinant E. coli strains were basically the same. The expression level of formate dehydrogenase in the latter was 342 IU/mL, which was significantly higher than 196 IU/mL in the former. In the 50 mL reaction system, 200 mM L-threonine was reacted at 220 rpm and 30 ℃ for 8 hours, the yield of L-ABA was 71% in the former and 85% in the latter. The results showed that increasing the expression level of formate dehydrogenase could significantly improve the synthesis efficiency of L-ABA. After the reaction temperature was optimized, the yield of L-ABA reached 90% catalyzed at 220 rpm and 30 ℃ for 8 hours. This study laid a solid theoretical foundation for the large-scale conversion and value-added of L-threonine and the biosynthesis of L-ABA in the future.