The steady state amperometric measurements of free cholesterol were performed by an enzyme electrode which was developed through electrostatic immobilization of the cholesterol oxidase in poly(vinylferrocenium) perchlorate, PVF+ClO4-, film that was coated on a Pt electrode. Amperometric measurements were performed at an applied potential of 0.7 V versus SCE. The effects of enzyme concentration, polymer film thickness, working temperature, dissolved oxygen gas and pH of solution on the response electrode were elucidated. The activation energy and the apparent Michaelis-Menten constant were calculated for enzyme electrode system. The linear working range, the sensitivity and the stability of the biosensor were also determined. The mechanism for the operation of the enzyme electrode was proposed. (c) 2006 Elsevier Inc. All rights reserved.