This paper describes a new amperometric biosensor for glucose monitoring. The biosensor is based on the activity of glucose dehydrogenase (GDH) and diaphorase (DI) co-immobilized with NAD + into a carbon nanotube paste (CNTP) electrode modified with an osmium functionalized polymer. This mediator was demonstrated to shuttle the electron transfer between the immobilized diaphorase and the CNTP electrode, thus, showing a good electrocatalytic activity towards NADH oxidation at potentials around +0.2V versus Ag|AgCl, where interfering reactions are less prone to occur.The biosensor exhibits a detection limit of 10μmolL −1 , linearity up to 8×10 −4 molL −1 , a sensitivity of 13.4μAcm −2 mmol −1 L −1 , a good reproducibility (R.S.D. 2.1%, n=6) and a stability of about 1 week when stored dry at 4°C.Finally, the proposed biosensor was applied for the determination of glucose in different samples of sweet wine and validated with a commercial spectrophotometric enzymatic kit.