This paper presents a novel biosensing platform for cholesterol accomplished by the spin-coating deposition of neutral polyaniline on poly(methyl methacrylate) support and the subsequent covalent immobilization of cholesterol oxidase onto polyaniline surface. The polyaniline film entraps the enzyme and it also acts as an active layer for optical sensing of free cholesterol. The effects of immobilization process of the enzyme over the oxidation state of polyaniline backbone and the optimum pH and temperature conditions for immobilized cholesterol oxidase were studied. The sensibility of the system to cholesterol in concentrations equivalent to those present in human serum evidences its potentiality in an optical method simply based on chemochromic properties of polyaniline. © 2013 Springer Science+Business Media Dordrecht.