Codon usage and rare codons have mixed results on the protein structure and function. An increasing amount of data is shown that replacing the rare codons with frequently synonymous ones has diverse results as a decrease in a protein’s specific activity, changing the folding pathway, and reducing protein solubility. In this study, we investigated the situation of codon usage of the Lampyridae family luciferases using computational databases. For this, the codon feature of these luciferases was studied, bioinformatically. Also, in silico analyses of this enzyme were conducted by structural modeling on the I-TASSER web server. The status of these rare codons in these structural models was studied using SPDBV and PyMOL software. Finally, the binding site properties were studied using the AutoDock Vina. Using molecular modeling, two rare codons (Arg533 and Arg536) were analyzed that may have a critical role in the structure and function of these luciferases. AutoDock Vina was used in molecular docking that recognizes some residues that yield closely related to luciferyl-adenylate binding sites. These analyses created a new understanding of the sequence and structure of these luciferases, and our findings can be used in some fields of clinical and industrial biotechnology. This bioinformatics analysis plays an essential role in the design of new drugs.