Here we report the synthesis, characterization and application of calixarene (Calix) modified montmorillonite (Mt) as a platform for bio-applications such as biomolecule immobilization and biosensing technologies. This modification enhanced the biomolecule immobilization capability of Mt. Initially, amino-functionalised calixarenes (Calix-NH2) were synthesized and used as a modifier. X-ray diffraction, Fourier transform infrared spectroscopy, zeta potential and thermal gravimetric analysis were performed to verify the modification of the clay minerals. For the biosensor construction, Calix-NH2 modified Mt (Calix-NH2/Mt), bovine serum albumin (BSA), glutaraldehyde (GA) and pyranose oxidase were immobilized on the surface of a glassy carbon electrode which was then referred to as a CalixNH(2)/ Mt/PyOx biosensor. After optimization of the enzyme amount and pH, analytical characteristics were investigated in detail.