An improved amperometric glucose biosensor was constructed by immobilizing glucose oxidase (GOD) in a titania sol-gel film, which was prepared by a vapor deposition method, on a Prussian Blue (PB)-modified electrode. The method combined the merits of immobilizing biomolecules in the titania sol-gel film by vapor deposition method and the synergic catalysis effects of PB and GOD molecules. Results showed that the fabricated titania sol-gel/PB membrane possessed high surface area, good mechanical stability, and good hydrophilicity, which provided a biocompatible microenvironment for maintaining the bioactivity of the immobilized enzyme and prevented the enzyme from leaking out of the film. Therefore, the present biosensor exhibited fast response time (10 s), high sensitivity (12.74 muA cm(-2) mM(-1)), long-term operational stability, good suppression of interference, and a wide linear range from 0.02 to 15 mM with a low detection limit of 5 muM for the detection of glucose. In addition, this simple and controllable method could fabricate biosensors in batches with a very small amount of enzyme.