2-Heptyl-4,7-di(thiophen-2-yl)-1H-benzo[d]imidazole) (BImTh) was synthesized. Electrochemical copolymerization of this monomer with 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)acetic acid (Fmoc-Gly-OH) was achieved on a graphite electrode and used as a matrix for amperometric cholesterol biosensing studies. In order to prepare a new cholesterol biosensor, cholesterol oxidase (ChOx) was covalently immobilized onto the copolymer coated graphite electrode. Cholesterol was used as the substrate and the decrease in oxygen level as a result of enzymatic reaction was monitored at -0.7V vs Ag reference electrode in a phosphate buffer (50 mM, pH 7.0). Kinetic parameters, storage stabilities and surface characteristics were investigated. K-M(app), I-max, LOD and sensitivity were calculated as 6.25 mu M, 9.69 mu A, 0.17 mu M and 2.47 mA/mM cm(2), respectively. This biosensor was applied to the determination of total cholesterol in serum samples. The estimation of cholesterol is an important for the diagnosis and prevention of several heart diseases and arteriosclerosis. Hence, it is important to develop new cholesterol biosensors. (C) 2013 Elsevier B.V. All rights reserved.