A nonenzymatic glucose fuel cell directly oxidizes glucose to gluconic acid as a Pt‐based abiotic catalyst in a proton exchange membrane environment and has various advantages (e. g., biocompatibility, chemical stability, high ionic conductivity). We report the permeability of a less hydrophobic diffusion layer for three flow‐field designs (serpentine, interdigitated, and parallel). In all cases, as the channel width of the flow path increases, the power density increases and the Ohmic resistance decreases. The serpentine shape (channel and rib widths: 1.5 and 0.5 mm, respectively) exhibits remarkable maximum power and current densities (cell voltage: 60 mV) of 103.4 μW cm−2 and 1,273 μA cm−2 compared to those of the parallel (46.5 μW cm−2 at 683 μA cm−2) and interdigitated (74.0 μW cm−2 at 878 μA cm−2) shapes, respectively. Furthermore, permeability and performance analyses according to the single‐cell temperature, glucose concentration, and flow rate changes provide various perspectives on glucose fuel cells.