Herein, an amperometric glucose oxidase (GOx) biosensor is presented using calixarenes as an immobilization matrix of the biomolecule. Firstly, thiol-containing calixarenes (Calix-SH) were synthesized, then self-assembled monolayers (SAMs) of Calix-SH on a gold surface were formed and hydroxyl groups of Calix-SH were activated using 1,1′-carbonyldiimidazole (CDI) chemistry. To test the usability of Calix-SH modified surfaces as a biosensor, glucose oxidase was used as a model biological component. After optimization of preparation and working conditions, our results indicate that the Calix-SH/GOx biosensor has a linear range in the range 0.1-1.0 mM (LOD: 0.015 mM) for glucose with a 25 s response time. Finally, the application of the biosensor was examined to detect glucose in real samples. The glucose amounts were calculated as 19.460 ± 0.521 and 31.647 ± 2.125 mM in coke and fizzy drink (with orange), respectively. To confirm the reliability of the Calix-SH/GOx biosensor, the calculated glucose concentrations which were analyzed by the Calix-SH/GOx biosensor were compared to conventional spectrophotometric glucose kits. The glucose amounts in coke and fizzy drink were calculated as 18.509 ± 0.732 mM and 31.579 ± 4.466 mM, respectively. © 2014 the Partner Organisations.