A stable three-dimensional glucose/oxygen enzymatic biofuel cell is fabricated based on the method of polymer encapsulation-based immobilization. And three-dimensional carbon felt is used as the substrate of the bio-electrode for increasing enzymatic loading density. Gold nanoparticles and multi-wall carbon nanotubes are employed to promote direct electron transfer and enhance conductivity and electron conduction rate of bio-electrodes. Glucose dehydrogenase and bilirubin oxidase are immobilized with tetrabutylammonium bromide (TBAB) modified Nafion, which enhances the stability of the bio-electrodes by the immobilization method. A membrane-free glucose/oxygen biofuel cell is assembled with a high open-circuit voltage of 0.85 V and a maximum power density of 21.9 ± 0.1 μW/cm2 in 0.1 M pH 7.0 phosphate buffer solution with 100 mM glucose and air saturation. And the biofuel cell shows high stability to the condition. After 60 days of periodic storage experiments, the performance of the enzymatic biofuel cell still maintained 90.3% of its electrochemical performance.