In this study, a novel amperometric glucose biosensor based on a selenium comprising conducting polymer and calixarene was developed. Firstly, poly(2-(2-octyldodecyl)-4,7-di(selenoph-2-yl)-2H-benzo[d][1,2,3]-triazole), poly((SBTz)) was electrodeposited onto a graphite electrode by an electropolymerization technique. Then, a newly synthesized calixarene and gold nanoparticle (AuNP) mixture was used for the improvement of biosensor characteristics. GOx, as a model enzyme was immobilized on the modified electrode surface. The constructed surface serves as a sufficient immobilization platform for the detection of glucose. Calixarenes and their derivatives may be a favouring agent for enzyme immobilization due to their specific configurations. Moreover, through the covalent binding between the carboxylic groups of the calixarenes and amino groups of the biomolecule, effective enzyme immobilization can be achieved while protecting the well-ordered structure of the enzyme molecule. Amperometric detection was carried out following oxygen consumption at -0.7 V vs. the Ag reference electrode in phosphate buffer (50 mM, pH 6.5). The proposed biosensor showed a linear amperometric response for glucose within a concentration range of 0.005 to 0.5 mM (LOD: 0.004 mM). K-m(app) and sensitivity were calculated as 0.025 mM and 102 mu A mM(-1) cm(-2), respectively. Scanning Electron Microscopy (SEM) was used to investigate the surface morphologies of successive modifications. Finally, the constructed biosensor was tested successfully to detect glucose in beverage samples.