Cholesterol is an essential structural component of mammalian cells, although elevated concentrations of cholesterol in the human body are linked to atherosclerotic disease. In this context, monitoring physiological cholesterol concentrations is of great interest in medical fields and the concept of producing electrical energy from cholesterol is interesting. In this work, we report the bioelectrocatalytic oxidation of cholesterol with the use of nicotinamide adenine dinucleotide-(NAD) dependent cholesterol dehydrogenase (ChDH) as an alternative enzyme to the commonly-used cholesterol oxidase (ChOx) in bioelectronic applications. To achieve bioelectrocatalytic cholesterol oxidation, ChDH was combined with diaphorase (DH) in a bilayer fashion, whereby DH acts as an intermediary enzyme to facilitate NADH oxidation at a ferrocene redox polymer. Characterization of the resulting bioanode identifies a linear cholesterol response range from 5 mu M to 200 mu M, with an associated sensitivity of 60.12 mA cm(-2) M-1. Furthermore, an O-2-reducing biocathode incorporating bilirubin oxidase (BOx) was combined with the cholesterol bioanode into a complete cholesterol/O-2 membrane-less enzymatic fuel cell (EFC). (C) The Author(s) 2016. Published by ECS. All rights reserved.