The electropolymerization of a laponite nanoparticle-amphiphilic pyrrole derivative-enzyme mixture preadsorbed on the electrode surface provides simultaneously the immobilization of the enzyme and the laponite particles in the polypyrrolic matrix. These incorporated laponite nanoparticles greatly enhance the sensitivity and stability of a cholesterol oxidase-based biosensor. Compared to a similar biosensor without laponite, the biosensor sensitivity increased from 5.1 to 13.2 mA M - 1 cm - 2 . Furthermore, the presence of hydrophilic laponite additive in the polymeric matrix containing cholesterol oxidase and cholesterol esterase is essential for the successful determination of total cholesterol.