In this study, we investigated the effect of mutations in orthogonal aminoacyl-tRNA synthetase (aaRS) on the incorporation efficiency of para-azido-L-phenylalanine (AzF), and performed molecular modeling. We targeted three amino acid positions (178, 248, and 256) in the aaRS cloned in the previously reported pEVOL-pAzF-C1-K776 and generated aaRS mutants (M178T, F248L, K256E) using site-directed mutagenesis. Subsequently, we transformed Escherichia coli DH10B (pSEVA631pt-sfGFP204amb) with each of the pEVOLpAzF-C1 plasmids containing the mutant aaRS and compared the specific fluorescence intensity upon AzF addition. With AzF addition, M178T and F248L mutants exhibited 1.36- and 1.19-fold increases, respectively, in specific fluorescence intensity compared to the control, while K256E mutant showed a 0.46-fold decrease. The selectivity for AzF was 1.987 for M178T, 1.931 for F248L, and 1.619 for K256E, all lower than the control. To interpret these experimental results, modeling and docking using Swiss-Model were performed. The results obtained in this study could be utilized for the development and interpretation of various aaRS for incorporating non-canonical amino acids into proteins.