A mediated whole cell biosensor was designed and characterized using glucose as the substrate. Gluconobacter oxydans cells were immobilized on the graphite electrode via sol-gel (tetraethyl orthosilicate)/chitosan hybrid composite modified with gold nanoparticles (TEOS/CHIT/AuNPs/G. oxydans). A soluble mediator, potassium hexacyanoferrate (III), was used for the amperometric detection of the respiratory activity of the intact bacterial cells. After optimization of preparation conditions of the TEOS/CHIT/AuNPs/G. oxydans biosensor, characterization studies have been carried out. The linear range for glucose was found to be 0.5-2.0 mM with a response time of 80 s. Moreover, repeatability and electrode-to-electrode reproducibility of the system were investigated and relative standard deviations were calculated as 1.7% and 2.2% for 1.0 mM glucose (n=5). Finally, the proposed system was applied to ethanol and glucose detection in real samples and the results were compared with a reference method.