An amperometric biosensor for determination of Cytochrome c (Cyt c) was fabricated by immobilizing Cytochrome c oxidase (COx) onto nickel oxide nanoparticles (NiO-NPs) decorated carboxylated multiwalled carbon nanotubes/polyaniline (NiO-NPs/cMWCNT/PANI) film electrodeposited on the surface of a gold (Au) electrode. The electrochemical characteristics of immobilized COx were investigated 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 of construction of enzyme electrode demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H2O2, which offers a number of attractive features to develop amperometric biosensors based on split of H2O2. There was a good linear relationship between the current (mA) and Cyt c concentration in the range 5 x 10(-12) M to 5 x 10(-7) M. The sensor had a detection limit of 5 x 10(-12) M (S/N = 3) with a high sensitivity of 3.7 mA cm(-2) nM(-1). The sensor gave accurate and satisfactory results, when employed for determination of Cyt c in different serum samples.