2,5-Dimethylpyrazine (2,5-DMP) is a building block for the pharmaceutical intermediate 5-methylpyrazine-2-carboxylic acid, and is also an important flavor compound in food. The chemical synthesis method has the disadvantages of poor selectivity, difficult to separate and purify the product, so it is not suitable for large-scale industrial production. In order to solve the above drawbacks and achieve high efficiency green production of 2,5-DMP, the l-threonine metabolic pathway in Escherichia coli (E. coli) was reconfigured by overexpressing l-threonine dehydrogenase (L-TDH) from E. coli and NADH oxidase (NOX) from Enterococcus faecium with the help of pACYCDuet-1 expression system. The EcTDH and EfNOX activities of the recombinant E. coli BL21(DE3)/pACYCDuet-1-Ectdh-Efnox was 23.55 and 2.83 IU/mL, respectively. And it can convert l-threonine to obtain 2,5-DMP with a yield of 2009.31 mg/L. Therefore, these researchs established solid practice foundation for future large-scale bioconversion of l-threonine to 2,5-DMP.