The retention, diffusion and structural properties of 2–4 nm nanoporous alumina membranes were investigated in view of their integration as size-selective interface in a glucose sensor. These membranes exhibit remarkable glucose diffusion properties with only a 5-fold reduction compared to free diffusion in water. The retention of the glucose-binding protein (Concanavalin A), which is characterized by a hydrodynamic radius of only 3.3 nm, was almost complete during at least 35 days. This high selectivity was also confirmed by SEM pictures analysis showing a highly uniform pores size distribution. Finally, the glucose sensor including a nanoporous membrane as size-selective interface was able to measure glucose levels in physiological solution during 25 days, which confirms that annealed alumina membranes are well suited for size-selective interface of biosensors.