A novel poly(vinyl alcohol) grafting 4-vinylpyridine self-gelatinizable copolymer was adapted to immobilize glucose oxidase. The reduction of hydrogen peroxide (H₂O₂) was detected at a Prussian Blue (PB) modified graphite electrode. A stable and sensitive glucose amperometric biosensor is described. The copolymer is a good biocompatible polymer in which the glucose oxidase retains high activity. Moreover, the copolymer can adhere firmly to the inorganic PB membrane. The sensor showed an apparent Michaelis–Menten constant of 18 ± 0.2 mM and a maximum current density of 1.14 µA cm^–2 mM^–1. The linear range is from 5 µM to 4.5 mM glucose and the detection limit is 0.5 µM glucose. The catalytic efficiency of PB for the reduction of H₂O₂ is higher than that for the oxidation of H₂O₂. Glucose concentrations in serum samples from healthy persons and diabetic patients were determined using the sensor. The results compared well with those provided by the hospital using a spectroscopy method.