A simple and robust cholesterol biosensor was designed by immobilizing cholesterol oxidase (ChOx) onto a conducting polymer modified graphite electrode. For this purpose, monomer, (Z)-4-(4-(9H-carbazol-9-yl) benzylidene) 2 (4 nitrophenyl) oxazol-5(4H)-one (CBNP), was synthesized and electrochemically polymerized on an electrode to achieve an effective immobilization platform for enzyme immobilization. After electropolymerization of the monomer (CBNP), electrochemical and spectroelectrochemical properties were investigated. Through the presence of nitro group on the polymer backbone hydrogen-bonding between enzyme molecules and polymer was achieved. Moreover, strong pi-pi stacking between aromatic moities in the polymer and aromatic residues of the enzyme enables a sensitive and reliable biosensor by conserving the crucial structure of biological molecules during the enzymatic reaction. The efficient interaction of the enzyme with the polymer coated surface brings easy and long-life detection of the substrate, cholesterol. After successful immobilization of ChOx with the help of glutaraldehyde as the crosslinking agent, amperometric biosensor responses were recorded at -0.7 V vs Ag wire in phosphate buffer (pH 7.0). K-M(app) (37.3 mu M), I-max (3.92 mu A), LOD (0.4063 mu M) and sensitivity (1.49 pA mu M-1 cm(-2)) values were determined. Finally, the prepared biosensor was successfully applied for determination of cholesterol content in real blood samples. (C) 2015 Elsevier B.V. All rights reserved.