A novel cholesterol biosensor was fabricated on hydrophobic ionic liquid (IL)/aqueous solution interface. The hydrophobic IL thin film played a signal amplification role because it not only enriched the cholesterol from the aqueous solution, but also immobilized matrix for cholesterol oxidase (ChOx). Prussian blue (PB) as advanced sensing materials was used as effective low-potential electron transfer mediation toward hydrogen peroxide (H2O2). The fabricated IL-ChOx/PB/Glassy carbon electrode (GCE) was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammogram (CV), respectively. And it exhibited a linear response to cholesterol in the range of 0.01-0.40 mM with a detection limit of 4.4 mu M. In addition, the kinetics behavior of cholesterol at IL-Chox/PB/GCE electrode was examined, and the electrocatalytic mechanism was proposed as shown in first scheme. ChOx immobilized in hydrophobic IL thin film was used as the first electrocatalyst for the cholesterol into H2O2, and the PB film onto the GCE was used as the second electrocatalyst for the 2e(-) reduction of the produced H2O2 into H2O. (C) 2012 Elsevier Ltd. All rights reserved.