It is necessary to apply a nonenzymatic glucose fuel cell using a proton exchange membrane for an implantable biomedical device that operates at low power. The permeability of glucose with high viscosity and a large molecular weight in the porous medium of the diffusion layer was investigated for use in fuel cells. Carbon paper was prepared as an anode diffusion layer, and it was analyzed with a diffusion layer treated with polytetrafluoroethylene (PTFE) and a microporous layer (MPL). When untreated carbon paper was applied, the peak power density (PPD) and open-circuit voltage (OCV) increased as the glucose concentration and flow rate increased. On this occasion, the highest PPD of 17.81 μW cm-2 was achieved at 3 mM and a 2.0 mL min-1 glucose aqueous solution (at atmospheric pressure and 36.5 °C). The diffusion layer, which became more hydrophobic through PTFE treatment, adversely affected glucose permeability. In addition, the addition of an MPL decreased OCV and PPD with increasing glucose concentrations and flow rates. Compared with untreated carbon paper, the PPD was six times lower approximately. Consequently, it was confirmed that the properties of carbon paper, such as low hydrophobicity, high porosity, and thin thickness, would be advantageous for nonenzymatic glucose fuel cells.