Anode catalyst layers for the direct glucose fuel cells (DGFC) were prepared with electrospray deposition method (ESD). Biofuel has attracted attention as a new energy source. However, the DGFC produced low power densities. The ESD enabled to deposit nanoparticles of catalysts and increase the surface area of them. In preparation of catalyst layers, the ratio of ionomer to carbon is shown as an important factor determining the performance of fuel cells. Although fuel flow rates also have impacts on the performance, the optimum value still remains unknown in DGFC. This study investigated the influence on the ionomer carbon ratio and the fuel flow rates to improve the generated power. We obtained the maximum power density of 9.85 mW/cm2 at an I/C ratio of 0.3 and an anode flow late of 10 ml/min. This value exceeds the performances of the previous study.