Since cholesterol triggers many diseases, many methods have been developed for its determination. In this study, an alternative system for cholesterol determination was developed by preparing amperometric biosensors. In the development of the biosensor, Pt/polypyrrole-polystyrene sulfonate film was prepared by electropolymerization of polypyrrole in polystyrene sulfonate medium using platinum surfaces. The cholesterol oxidase enzyme was immobilized on the prepared platinum/polypyrrole-polystyrene sulfonate electrode. For molecular determination in the prepared cholesterol biosensor, a series of enzymatic reactions were performed on the enzyme electrode surface at +0.40 V by utilizing the oxidation of hydrogen peroxide. The effects of environmental conditions such as temperature and pH that affect the performance of the biosensor were investigated, and the most suitable conditions for the biosensor were determined. The linear working range of the amperometric biosensor for cholesterol determination was determined. In enzyme immobilization, calculations were made for the Michaelis-Menten constants K