Microporous layer (MPL) is a key component in the management of water (and to a lesser degree, heat) for enhanced performance, reliability and durability of proton exchange membrane (PEM) fuel cells at high current densities for a wide range of commercial applications. In this study, as an alternative to conventional nanomaterials, such as Ketjenblack, graphene, a monolayer of carbon atoms arranged in a two-dimensional lattice, is considered to be an ideal MPL material due to its unique characteristics, including excellent electrical and thermal conductivity. A graphene-based MPL has been prepared and investigated for its effective water management and performance enhancement for PEM fuel cells through morphological, structural, physical and electrochemical characterization and performance testing for single large cells. Comparison studies are also conducted with the MPL made of conventional material, Ketjenblack. It is shown that the graphene-based MPL demonstrates comparable performance to the one made of Ketjenblack under high-humidity operation, while exhibiting excellent performance superiorities (up to a peak power density improvement of approximately 55%) under low-and intermediate-humidity operation. Overall, the graphene-based MPL has significant potential to meet performance demand under a wide range of operating conditions.