Monitoring the cholesterol level is of great importance, especially for people with high risk of developing heart disease. Here we report on reagentless cholesterol detection in human plasma with a novel single-enzyme, membrane-free, self-powered biosensor, in which both cathodic and anodic bioelectrocatalytic reactions are powered by the same substrate. Cholesterol oxidase was immobilized in a sol-gel matrix on both the cathode and the anode. Hydrogen peroxide, a product of the enzymatic conversion of cholesterol, was electrocatalytically reduced, by the use of Prussian blue, at the cathode. In parallel, cholesterol oxidation catalyzed by mediated cholesterol oxidase occurred at the anode. The analytical performance was assessed for both electrode systems separately. The combination of the two electrodes, formed on high surface-area carbon cloth electrodes, resulted in a self-powered biosensor with enhanced sensitivity (26.0 mA M(-1) cm(-2)), compared to either of the two individual electrodes, and a dynamic range up to 4.1 mM cholesterol. Reagentless cholesterol detection with both electrochemical systems and with the self-powered biosensor was performed and the results were compared with the standard method of colorimetric cholesterol quantification.