An amperometric biosensor based on Pt electrodes modified with a thin film of a Ni, Al layered double hydroxide (LDH), submitted to a preliminary oxidative treatment in order to have the nickel centers at the oxidation state þ 4, and glucose oxidase (GOx) is presented. The oxidized LDH acts both as a system to support the enzyme and as a barrier to anions since it acquires an overall negative charge, as demonstrated by electrochemical impedance spectroscopy. Even if the biosensor response is due to the detection of H 2 O 2 at anodic potentials, glucose can be accurately determined in the presence of ascorbic acid or other interferences, commonly present in real matrices in anionic form, since they can not reach the electrode surface. The effectiveness of the developed biosensor has been demonstrated by measuring glucose in samples of fruit juices containing ascorbic acid at high levels.