Conventional catalyst layer with a smooth surface exists the larger area of“catalytic dead zone” and reduces the utilization of catalyst. Based on this, a novel catalyst layer structure based surface-patterned Nafion® membrane was designed to achieve more efficient electrochemical reaction in this work. Surface-patterned Nafion® membranes were prepared by hot pressing with different pressures, and their swelling degrees reduced obviously with the increase of pressure, but proton conductivities of the membranes were almost unchanged. Pre-swelling and direct-spraying deposition methods were used to prepare the novel catalyst layer, and the effect of pressure on the performance of MEA was investigated. The results suggested that the peak power density of DMFC with optimal novel catalyst layer structure increased by 28.84%, the charge transfer resistances of anode and cathode reduced by 28.8% and 26.5% respectively, compared with the conventional catalyst layer. Performance improvement is attributed to the fact that the novel catalyst layer structure optimizes the electrolyte membrane/catalyst layer and gas diffusion layer/catalyst layer interfacial structure, which increases the electrochemical reaction region and reaction sites. The novel catalyst layer with a three-dimensional curved surface structure enlarges the “three-phase boundaries (TPB)” and electrochemical active surface area (ECSA) of membrane electrode assembly (MEA). Therefore, this work provides an effective solution to achieve the high performance of DMFC by optimizing the internal interface structure of electrode, which is helpful to the future development of DMFC.