Clay based biosensors were developed using montmorillonite (Mont) modified with methyl (M) and dimethylamine (DM). X-ray diffraction, Fourier transform infrared spectroscopy, zeta potential and thermal gravimetric measurements were used to characterize the modified clays. After immobilization of glucose oxidase (GOx) via clay on the glassy carbon electrode, its application as a glucose biosensor was investigated in detail. The best response characteristics were obtained by DM-Mont and optimization of enzyme amount, reproducibility of biosensor fabrication, repeatability of measurements and operational stability were all evaluated. The optimized biosensor showed a very good linearity between 0.05 mM and 1.0 mM, a 7 s response time and a limit of detection to glucose of 0.038 mM. Also, kinetic parameters and stabilities were determined. Apparent K-m and I-max values were found as 0.73 mM and 2.955 mu A, respectively. As well as batch configuration, the DM-Mont/GOx biosensor was successfully applied in the flow injection analysis mode. Finally, the performance of the DM-Mont/GOx biosensor to analyze glucose in a wine sample was compared with HPLC.