The convergence of biotechnology and nanotechnology has led to the development of hybrid nanomaterials that incorporate the highly selective catalytic and recognition properties of biomaterials. The conjugation of nanoparticles (NPs) and other nanoobjects (e.g. nanorodes, nanowires and nanotubes) with biomolecules is an attractive area of research within nanobiotechnology. Here, this study focused on the design of cytochrome c/nickel oxide nanoparticles/glassy carbon electrode, prepared by the electrochemical deposition of the nickel oxide nanoparticles (NiO NPs) on the glassy carbon (GC) electrode surface and the self-immobilization of cytochrome c on the surface of the electrodeposited nickel oxide nanoparticles. The existence of different geometrical shapes of the NiO NPs was exhibited using the scanning electron microscopy (SEM) and atomic force microscopy (AFM). These geometrical structures could lead to the better immobilization of proteins on their surfaces. The resulting electrode displayed an excellent behavior for the redox of the cytochrome c. Also, the resulting heme protein exhibited a direct electrical contact with the electrode as a result of the structural alignment of the heme protein on the nanometer-scale nickel oxide surfaces. In line with our investigation, non-physiological electrochemical responses were analyzed for this electrode.