When inadequate water purification and sewage disposal systems, cholera poses a significant health threat in developing nations, Vibrio cholerae (V. cholerae) is one of the mindful microscopic organisms associated with cholera illness. If cholera is not treated, renal failure, shock, hypokalemia, and pulmonary edema can occur, resulting in death in a matter of hours. The machinery of bacterial virulence factors is what causes this disease. Among the various V. cholerae strains, V. cholerae O1 is the most prevalent and pathogenic strain. The total genome succession of V. cholerae unravels the presence of different genes and uncharacterized proteins whose capabilities are not yet perceived. Therefore, it is essential to comprehend V. cholerae by analyzing the structure and annotating the function of uncharacterized proteins. The NCBI sequence of uncharacterized V. cholerae O1 EET91795.1 proteins has been annotated for this study. Domain family, protein solubility, ligand binding sites, and other parameters have all been determined using a variety of databases and computational tools. The protein's ligand-binding sites were found, and its three-dimensional structure was modeled. According to the analysis, the hotdog family protein may play metabolic roles like thioester hydrolysis in the metabolism of fatty acids and the breakdown of two products such as phenylacetic acid and the pollutant 4-chlorobenzoate. The structural prediction of this protein and detection of binding sites would suggest a potential target to uncover promising inhibitors against the protein to treat infection caused by the target strain.