Cholesterol oxidase (ChOx) physisorbed onto NiO nanoparticles (nNiO, 22 nm)— chitosan (CHIT) film prepared using coprecipitation method has been characterized using x-ray diffraction, Fourier transform infrared, scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy techniques. The results of electrochemical response studies conducted on ChOx/nan-NiO-CHIT/ITO bioelectrode show linearity of 10–400 mg/dl, detection limit of 43.4 mg/dl, sensitivity of 0.808 μA/(mg dl cm2), fast response time of 15 s, and shelf-life of about 10 weeks. The low value of Michaelis–Menten constant (Km) obtained as 0.67 mM indicates high affinity of ChOx toward the substrate.