The non-natural amino acid 4-nitrophenylalanine is a crucial pharmaceutical ingredient and has extensive utility in protein engineering. Here, we demonstrated the production of 4-nitrophenylalanine by Escherichia coli with AurF, 4-aminobenzoate N-oxygenase from Streptomyces thioluteus. Firstly, eight distinct gene combinations, encompassing four variants of papA and two of papBC, were evaluated to optimize the production of 4-aminophenylalanine, a precursor of 4-nitrophenylalanine. The strain co-expressing both pabAB from E. coli and papBC from Streptomyces venezuelae attained the highest 4-aminophenylalanine production. In a fed-batch fermenter cultivation, 4-aminophenylalanine production of 22.5 g/L was achieved. To produce 4-nitrophenylalanine from glucose, we constructed strains co-expressing AurF alongside the genes responsible for 4-aminophenylalanine synthesis. The subsequent optimization of the plasmid copy numbers carrying each gene set resulted in an increase in the 4-nitrophenylalanine production titer. Transcription analysis revealed that the expression level of the 4-aminophenylalanine biosynthetic genes markedly contributed to 4-nitrophenylalanine production. After optimizing batch fermentation conditions, the titer of 4-nitrophenylalanine increased to 2.22 g/L. Overall, these results provide the basis for industrial microbial production of 4-nitrophenylalanine, contributing to the advancement of biotechnological methodologies for generating non-natural amino acids with specific functionalities.