Abstract: Pyridoxine 5'-phosphate oxidase (PdxH) is a rate-limiting enzyme in the biosynthesis pathway of pyridoxal-5'-phosphate (PLP), which is the key to the efficient synthesis of PLP. PdxH from Nakaseomyces bracarensis (NbPdxH) was selected as the study object. NbPdxH was modified through directed evolution and high-throughput screening to improve the catalytic activity of the enzyme. A pRB1s-NbpdxH^M8/BW25113 mutant with improved catalytic efficiency was successfully obtained through screening. The PLP production reached 5.3 g·L⁻¹, and the conversion ratio of PNP was 71%, which was 27% higher than that of the original strain. Through the structural analysis, it was speculated that the charge near the active site of NbPdxH^M8 was changed, which improved the binding efficiency of the enzyme and the substrate. The relevant research results provided a new idea for the efficient synthesis of PLP.