A commercial strain of Gluconobacter oxydans together with a new co-polymer Poly(glycidyl methacrylate-co-3thienylmethylmethacrylate) (Poly(GMA-co-MTM)), which provides effective immobilization in the continuous flow system, was used in the sensor design. By taking the advantages of the nano-technology, carbon nanotubes (CNTs) were also added to the cell film and the sensitivity of the sensor was increased about 15 times. During the glycerol analysis in the continuous system, it was shown that composite film was not removed from the electrode surface and film elements were not washed out from the system. Glycerol analyses were performed by using batch loaded continuously flow cell at different flow rates of 1, 2, 4, and 6 mL/min. The linear range was found as 2-100 mM with the detection limit (LOD) of 0.057 mM according to S/N = 3. The calibration graphs were obtained for Poly(GMA-co-MTM)/G. oxydans and Poly(GMA-co-MTM)/CNT/G. oxydans biofilm electrodes in FIA mode, and sensitivities were found to be 1.50 nA/mM and 19.13 nA/mM, respectively. In this study, Poly(GMA-co-MTM) was used for the first time as a microbial matrix and was shown to be an effective immobilizatipn agent (C) 2015 Elsevier B.V. All rights reserved.