Preparation and characterization of a thin-film cholesterol biosensor employing an organic-inorganic hybrid system of cholesterol oxidase (ChOx) and Prussian blue (PB) is described. ChOx was immobilized in Langmuir-Blodgett (LB) films consisting of positively charged octadecyltrimethylammonium (ODTA) and nano-sized PB clusters. Immobilization was performed by simple immersion of ODTA/PB LB films into an aqueous solution of ChOx. Subsequent ChOx absorption into LB films was confirmed by infrared reflection-absorption spectroscopy. Obtained ODTA/PB/ChOx LB films clearly exhibited a response current to cholesterol under an applied potential of 0.0 V (vs. Ag/AgCl). The linearity of current density versus cholesterol concentration was confirmed for the range 0.2-1.2 mmol/L The present study indicates that simple immersion of ODTA/PB LB films into an enzyme solution is a promising method to produce many types of thin-film biosensors comprising a hybrid system of an oxidative enzyme and PB nano-clusters that work at a very low potential range. (C) 2009 Elsevier B.V. All rights reserved.