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In this study, an amperometric cholesterol biosensor was constructed based on cholesterol oxidase immobilized on a conducting 4-(4H-dithienol[3,2-b:2’,3’d]pyrrole-4)aniline polymer, (DTP(aryl)aniline). Glassy carbon electrodes were covered with P(DTP(aryl)aniline) which is used for the wiring of enzyme to the electrode surface by using electro-polymerization. The electron transfer was successfully made by the bio-catalytic activity and possession of the unique morphology of the polymer allowed efficient immobilization of the cholesterol oxidase enzyme. Analytical performances; linear range, detection limit, limit of quantification and the Michaelis-Menten constant (Km) of biosensor electrodes were obtained 2.0 mM–23.7 mM, 0.27 mM, 0.82 mM, 17,81 mM respectively. Biosensor optimization parameters: optimum pH, optimum temperature, stability test and response time were evaluated. The real sample and recovery studies were also performed in order to show applicability of the biosensing electrodes .