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The electrochemical performance of a glassy carbon electrode coated with polypirydyl dinuclear ruthenium oxo-complex [(bpy)2(NH3)RuIII(µ-O)RuIII(NH3)(bpy)2]4+ incorporated into perfluorosulfonated polymer membrane (Nafion®) was studied in aqueous solution (acetate buffer 0.1 mol L-1). Following the electrochemical analysis of the modified electrode, the enzyme glucose oxidase (Aspergillus níger) was immobilized by adsorption onto the surface of the coated electrode. The mediation of electron-transfer by oxo-bridged dinuclear ruthenium ammine [(bpy)2(NH3)RuIII(µ-O)RuIII(NH3)(bpy)2]4+ for the oxidation of glucose was investigated by cyclic voltammetry in acetate buffer and showed a single electron in the process of oxidation/reduction of the redox couple RuIII-O-RuIII/RuIII-O-RuIV. These ruthenium (III) complexes exhibit appropriate redox potentials of 0.131–0.09 V vs. SCE to act as electron-transfer mediators. The plot of anodic current vs. the glucose concentration of the biosensor was linear in the concentration range between 2.52 × 10-5 and 1.00 × 10-4 mol L-1. Moreover, the apparent Michaelis–Menten kinetic (KMapp) and the catalytic (Kcat) constants were 8.757 × 10-6 mol L-1 and 1,956 s-1, respectively, which agree well with values in the literature and demonstrate the efficiency of the ruthenium dinuclear oxo-complex [(bpy)2(NH3)RuIII(µ-O)RuIII(NH3)(bpy)2]4+ as a mediator of redox electron-transfer.