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A novel impedimetric biosensor was fabricated for total cholesterol sensing based on platinum nanoparticle and polypyrrole multilayer nanocomposite electrode. The Pt nanoparticles (PtNP) electrochemically deposited between two polypyrrole layers on indium tin oxide (ITO) glass plates (PtNP/PPY/ITO) have offered high-electroactive surface area and favourable microenvironment for immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) resulting in enhanced electron transfer between the enzyme system and the electrode. Impedimetric response studies of the ChEt-ChOx/PtNP/ITO nanobioelectrode exhibit improved linearity (2.5 × 10-4 to 6.5 × 10-3 M/l), low detection limit (2.5 × 10-4 M/l), fast response time (25 s), high sensitivity (196 Ω/mM/cm-2) and a low value of the Michaelis-Menten constant (Km, 0.2 M/l) with a regression coefficient of 0.997.