We report the results of studies relating to the in situ synthesis of a novel ternary NiFe2O4/CuO/FeO-chitosan nanocomposite, which could be utilized as a cholesterol biosensor. The phase identification, morphology and particle size of the NiFe2O4/CuO/FeO nanocomposite have been investigated via X-ray diffraction pattern (XRD), scanning electron microscopy (SEM), high resolution transmission electron microscope (HR-TEM) and Fourier transform infrared (FTIR) spectroscopy. The quantification of cholesterol was accomplished by immobilizing cholesterol oxidase (ChOx) onto a chitosan-NiFe2O4/CuO/FeO nanocomposite (NiFe2O4/CuO/FeO-CH NC) deposited onto an indium-tin-oxide (ITO) glass substrate via the sol-gel technique. The electrochemical study results of the biocompatible ChOx/NiFe2O4/CuO/FeO-CH/ITO electrode reveal good linearity (50-5000 mg/L), a low detection limit (313 mg/L), high sensitivity (0.043 mu A/(mg/L cm(-2))), a fast response time (10 s) and a shelf-life of 3 months. The low Michaelis-Menten constant (K-m) of 80 mg/L (0.21 mM) indicates the high affinity of ChOx for the analytes. Further, this bioelectrode has been used in clinical applications to estimate cholesterol levels with negligible interference (2%) from analytes present in human serum samples. (C) 2012 Elsevier Ltd. All rights reserved.