A new biosensor based on immobilization of glucose oxidase (Gox) using enzyme solution containing o-phenylenediamine(oPD) on platinum nanoparticles (PtNPs) electrodeposited polyvinylferrocenium perchlorate matrix (PVF +ClO4-) was fabricated for the bioelectrochemical determination of glucose. Firstly, PVF+ClO 4- film was electroprecipitated on Pt electrode by electrooxidation of polyvinylferrocene, and then PtNPs were electrodeposited on PVF+ClO4-/Pt. Finally, Gox was immobilized simultaneously with the electropolymerization of oPD on PtNPs/PVF +ClO4-/Pt. The biosensor showed excellent anti-interference ability to ascorbic acid and uric acid due to the permselectivity of PoPD in the structure of the proposed biosensor. The linear range of Gox-PoPD/PtNPs/PVF+ClO4-/Pt was from 0.06 mM to 9.64 mM with a sensitivity of 17.40 μA mM-1 cm -2 and a detection limit of 0.018 mM. The effects of applied potential, PVF+ClO4- film thickness, oPD concentration, Gox concentration, electropolymerization time and temperature on the response current of the biosensor were investigated in detail. The electrocatalytic effect of PtNPs as well as repeatability, reproducibility and stability of the biosensor were also studied. PtNPs modified PVF +ClO4-/Pt exhibited excellent electrocatalytic activity to the electrooxidation of H2O2. In addition, the biosensor was successfully applied for the determination of glucose concentration in human blood serum samples. © 2014 Elsevier Ltd.