Present study highlights the importance of ZnO matrix prepared by vapour phase transport technique on platinum coated Si platform (ZnO/Pt/Si) as a potential matrix for the realization of highly sensitive and selective bioelectrode for detection of total cholesterol. Bienzymes cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) have been immobilized onto the surface of ZnO thin film matrix by physical adsorption technique. The prepared bioelectrode (ChEt-ChOx/ZnO/Pt/Si) is utilized for detection of total cholesterol using the cyclic voltammetry technique. The bioelectrode (ChEt-ChOx/ZnO/Pt/Si) is found to exhibit efficient sensing response characteristics with high sensitivity of 190 mu A mM(-1) cm(-2); good linearity in the range of 0.5-12 mM total cholesterol concentration, and a very low Michaelis-Menten constant of 0.68 mM which indicates high affinity of bienzymes immobilized on ZnO towards the analyte (total cholesterol). The enhanced response is attributed to the development of ZnO thin film based matrix having good electron transport property and nanoporous morphology for effective loading of enzymes with favourable orientation. (C) 2014 Elsevier B.V. All rights reserved.