Cholesterol oxidase (ChOx) has been covalently immobilized onto electrophoretically deposited conducting polymer film (on indium-tin-oxide (ITO) glass plate) derived from nano-structured polyaniline (PANI) colloidal suspension using N-ethyl-N'-(3-dimethylaminopropyl) corbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry. These PANI/ITO and ChOx/PANI/ITO electrodes have been characterized using ultraviolet-visible (UV-vis), Fourier transform-infrared (FT-IR), scanning electron microscopy (SEM), and impedance spectroscopy techniques, respectively. These ChOx/PANI/ITO bio-electrodes exhibit linearity from 25 to 400 mg dL(-1) of cholesterol, detection limit as 25 mg dL(-1) and sensitivity as 7.76x10(-5) Abs(mg/dL)(-1). The value of the apparent Michaelis-Menten constant (Km(app)) calculated from amperometric response studies has been found to be 0.62 mM for ChOx/PANI/ITO bio-electrode.