The determination of cholesterol is greatly important because high concentrations of this biomarker are associated to heart disease. Moreover, cholesterol can be used as a fuel in enzymatic fuel cells operating under physiological conditions. Here, we present a cholesterol biosensor and a peroxide-free biofuel cell based on the electrocatalytic oxidation of the NADH generated during the enzymatic reaction of cholesterol dehydrogenase (ChDH) as an alternative to the H2O2 biosensing strategies used with cholesterol oxidase-bioelectrodes. Azure A functionalized-carbon nanodots were used as NADH oxidation electrocatalysts and for ChDH covalent immobilization. The biosensor responded linearly to cholesterol concentrations up to 1.7 mM with good sensitivity (4.50 mA cm−2 M−1) and at a low potential. The ChDH bioelectrode was combined with an O2-reducing bilirubin oxidase cathode to produce electrical energy using cholesterol as fuel and O2 as oxidant. Furthermore, the resulting enzymatic fuel cell was tested in human serum naturally containing free cholesterol.