A novel amperometric glucose biosensor has been fabricated on the basis of aligned ZnO nanorod film grown on ITO directly. Glucose oxidase immobilized on the surface of ZnO nanorods are very stable with highly catalytic activity during the measurements, Because of the novel properties of ZnO, such as biocompatibility, non-toxicity, chemical stability, electrochemical activities and high isoelectric point, and the protection effect of Nifion membrane cast on the surface of the film. This biosensor displays excellent analytical performance over a wide linear range along with good selectivity. Interference from uric acid and ascorbic acid which usually coexist with glucose in practical samples has been found to be negligible. This method may be used to construct other amperometric biosensors using aligned nanorod/nanowire films.