High-level in vivo reactive oxygen species (ROS) can damage many biomolecules via oxidative stress and play vital roles in the pathogenesis of several bodily disorders. Therefore, fast and sensitive monitoring strategies for trace ROS, such as hydrogen peroxide (H2O2), are of great significance. Herein, we present a highly sensitive field-effect transistor (FET) sensor based on single-layer graphene for trace hydrogen peroxide (H2O2) detection. Graphene and cytochrome c (Cyt c) were employed as the conductive substrate material and biomolecular receptor for H2O2 detection, respectively. High-efficiency charge transfer can be achieved by reliable electrical contact across the Cyt c/underlying graphene interface. The Cyt c/graphene FET platform exhibited hole-transport behavior with high conductivity and high sensitivity toward H2O2 with a detection limit of 100 fM and rapid response time (<1 s). Moreover, our sensor platform was able to specifically discriminate H2O2 from a series of interfering substances, such as dopamine, ascorbic acid, glucose, uric acid and glutamate. This result, therefore, demonstrates that the proposed Cyt c/single-layer graphene FET sensor could facilitate the high-efficiency charge transfer between the redox center of the Cyt c/graphene interface, indicating a promising application in future trace H2O2 or free radical biosensors.