Our laboratory studies diffusion characteristics according to flow channel architecture and diffusion layer properties in operating a glucose fuel cell. Glucose is the most abundant monosaccharide in nature, theoretically has a high energy density per mass, and has various advantages (e.g., eco-friendliness, endogenous compounds in the body, non-toxic, non-flammable). Additionally, the nonenzymatic glucose fuel cell oxidizes glucose to gluconic acid by an abiotic catalyst, and the proton exchange membrane has advantages (e.g., mechanical durability, chemical stability, biocompatibility) compared to the anion exchange membrane. A nonenzymatic glucose fuel cell is constructed through the structural design and fabrication of the flow path plate and diffusion layer, and the performance of the fuel cell is evaluated through polarization curves and electrochemical impedance spectroscopy (EIS). Through optimized design and electrochemical analysis, we report the possibility of using a glucose fuel cell as an eco-friendly energy source.