An amperometric glucose biosensor was developed based on the immobilization of glucose oxidase in the organically modified silicate (ormosil)-polyvinyl acetate (PVA) matrix onto a Prussian Blue (PB)-modified glassy carbon electrode. A higher stability PB-modified electrode was prepared by the electrochemical deposition of FeCl 3 , K 3 [Fe(CN) 6 ] and ethylenediamine tetraacetic acid (EDTA) under cyclic voltammetric (CV)conditions. The effects of the potential range of CV conditions, electrolyte cations, applied potential, pH, temperature and co-existing substances were investigated. The detection limit of the glucose biosensor was 8.1 μmol·L −1 (S/N = 3) with a linear range from 20 μmol·L −1 to 2 mmol·L −1 (R = 0.9965). The biosensor presented a fast response and good selectivity. Additionally, excellent reproducibility and stability of the biosensor were observed.Prussian Blue, glucose oxidase, ormosil-PVA, ethylenediamine tetraacetic acid, biosensor