Here we report on the design and study of a biofuel cell consisting of a glucose oxidase-based anode (Aspergillus niger) and a laccase-based cathode (Trametes versicolor) using osmium-based redox polymers as mediators of the biocatalysts' electron transfer at graphite electrode surfaces. The graphite electrodes of the device are modified with the deposition and immobilization of the appropriate enzyme and the osmium redox polymer mediator. A redox polymer [Os(4,4′-diamino-2,2′bipyridine)2(poly{N-vinylimidazole})-(poly{N-vinylimidazole})9Cl]Cl (E0′ = −0.110 V versus Ag/AgCl) of moderately low redox potential is used for the glucose oxidizing anode and a redox polymer [Os(phenanthroline)2(poly{N-vinylimidazole})2-(poly{N-vinylimidazole})8]Cl2 (E0′ = 0.49 V versus Ag/AgCl) of moderately high redox potential is used at the dioxygen reducing cathode. The enzyme and redox polymer are cross-linked with polyoxyethylene bis(glycidyl ether). The working biofuel cell was studied under air at 37 °C in a 0.1 M phosphate buffer solution of pH range 4.4-7.4, containing 0.1 M sodium chloride and 10 mM glucose. Under physiological conditions (pH 7.4) maximum power density, evaluated from the geometric area of the electrode, reached 16 μW/cm2 at a cell voltage of 0.25 V. At lower pH values maximum power density was 40 μW/cm2 at 0.4 V (pH 5.5) and 10 μW/cm2 at 0.3 V (pH 4.4).