We describe a modification of glassy carbon (GC) electrode surface with electrodeposited cobalt oxide nanoparticles (CoOx NPs), and then, this electrode is used to immobilization of Cytochrome c (Cyt c). The morphology of electrodeposited CoOx NPs was analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Electrochemical redox property investigation of immobilized protein (Cyt c) molecules was accomplished by cyclic voltammetry (CV) technique. Cyt c/CoOx NPs/GC electrode exhibits electron transfer between the protein prosthetic groups and electrodeposited nanoparticles on the electrode surface and displayed a couple of stable redox peaks with a formal potential (EA degrees(')) of -89.5 mV (107.5 mV vs. NHE) with respect to reference electrode in 0.05 M phosphate buffer solution (pH 7.0). The experimental results demonstrated that Cyt c/CoOx NPs/GC biosensor exhibited electrocatalytic activity toward the reduction of hydrogen peroxide and application to hydrogen peroxide determination was exemplified.