The combination of supramolecules and conducting polymers (CPs) has gained much attention for the development of new immobilization matrices for biomolecules. Herein, an amperometric biosensor based on a novel conducting polymer, poly(2-(2-octyldodecyl)-4,7-di(selenoph-2-yl)-2H-benzo[d][1,2,3]triazole)) (PSBTz) and beta-cyclodextrin (beta-CD) for the detection of cholesterol, was constructed. The PSBTz film with beta-CD was deposited on a graphite electrode by electropolymerization technique to achieve a suitable matrix for enzyme immobilization. Moreover, to justify the immobilization, alkyl chain containing conducting polymer (PSBTz) was designed, synthesized and electrochemically polymerized on the transducer surface. Alkyl chains in the structure of SBTz and hydroxyl groups of beta-CD contributed to effective immobilization while protecting the suitable orientation of the biomolecule. Cholesterol oxidase (ChOx) was covalently immobilized onto the modified surface using N,N'-carbonyldiimidazole (CDI) as the cross-linking agent. After successful immobilization, amperometric biosensor responses were recorded at -0.7 V vs Ag/AgCl in phosphate buffer (pH 7.0). The apparent Michaelis-Menten constant (KMapp), maximum current (I-max), limit of detection (LOD), and sensitivity values were determined: 28.9 mu M, 12.1 mu A, 0.005 mu M, and 5.77 mu A/mu M cm(2), respectively. The fabricated biosensor was characterized using scanning electron microscopy (SEM) and cyclic voltammetry (CV) techniques. Finally, the prepared biosensor was successfully applied for the determination of cholesterol in blood samples.