Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) electrode. An amperometric H2O2 biosensor was constructed by connecting this modified Au electrode along Ag/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H2O2, which offered a number of attractive features to develop an amperometric biosensor based on split of H2O2. There was a good linear relationship between the current (mA) and H2O2 concentration in the range 3-700 mu M. The sensor had a detection limit of 0.2 mu M (S/N = 3) with a high sensitivity of 3.3 mA mu M-1 cm(-2). The sensor gave accurate and satisfactory results, when employed for determination of H2O2 in different fruit juices. (C) 2012 Elsevier Ltd. All rights reserved.