The key-point for the development of an amperometric sensor is the immobilization of the enzyme. In the present work we use biosensors based on glucose oxidase (GOD) onto electrodes nanostructured with carbon nanotubes (CNT), to be employed in cell culture monitoring. The goal is to determine the best immobilization strategy from the point-of-view of sensor lifetime. We compared three types of immobilization: the spontaneous adsorption of the enzyme on nanotubes, the entrapment in a Nafion matrix (optimizing also the quantity), and the cross-linking with glutaraldehyde. The cross-linking gives the best sensitivity, 17.38 μA mM^-1 cm^-2, and the lowest detection limit, 25 μM. On the other hand, Nafion matrix allows to extend the linear range up to 7.5 mM. Finally, electrodes are tested over 35 days to analyze the lifetime. GOD cross-linking results to have 100% of retained activity after 35 days, while the adsorption and the entrapment retain only the 20 % of the original response.