This study introduces the application of genetic code expansion in Escherichia coli, focusing on the stable integration of aminoacyl-tRNA synthetase (aaRS) and tRNA genes for the enhanced incorporation of non-canonical amino acids (ncAAs), specifically p-azido-L-phenylalanine (pAzF). Genomic integration was achieved through the lambda red system, which was able to overcome the limitations of previous conventional plasmid-based methods. The activity of the aaRS/tRNA pair expressed from the genome was confirmed by super-fold green fluorescence protein (sfGFP), which contained an amber stop codon. The integrated strain demonstrated enhanced site-specific pAzF incorporation over plasmid-based system, attributable to its selective incorporation. Our findings demonstrate a significant improvement of efficiency and stability in ncAA incorporation, thereby offering a promising method for large-scale protein production and synthetic biology applications.