Cholesterol oxidase (COX) is an essential enzyme involved in cholesterol oxidation, playing a vital role in various biochemical processes. This study focused on the molecular characterization and chemical engineering of COX derived from endophytic bacteria found in medicinal plants. The COX gene in these bacterial strains was successfully amplified and characterized using molecular methods such as polymerase chain reaction (PCR) and DNA sequencing. Analysis of the obtained sequences provided insights into the genetic diversity and phylogenetic relationships of the COX gene in these bacteria. The enzymatic properties of the purified COX enzyme, including substrate specificity, optimal conditions, and stability, were also investigated. These findings enhance our understanding of the catalytic potential and functional features of COX from endophytic bacteria in medicinal plants. Additionally, the study explored chemical engineering approaches to improve the enzymatic efficiency and stability of COX. Strategies like enzyme immobilization and protein engineering were employed to enhance the performance of COX. This chemical engineering aspect offers opportunities for the development of more efficient and robust COX-based biocatalysts. The molecular characterization and chemical engineering of COX from endophytic bacteria found in medicinal plants hold significant value. The insights gained not only expand our knowledge of the genetic diversity and enzymatic properties of COX but also lay the groundwork for potential applications in biotechnology and the pharmaceutical industry. Harnessing the catalytic capacity of endophytic bacteria's COX may lead to the creation of innovative therapeutic drugs and diagnostic tools for cholesterol-related disorders.