Hydrogen peroxide (H2O2) is an essential mediator for most of the oxidative biological reactions in enzyme-based biosensor systems, such as glucose oxidase, cholesterol oxidase, and alcohol oxidase. Synthesis of new catalysts to detect the concentration of H2O2 more precisely is indispensable for enzyme-based electrochemical biosensors. In this study, boron-doped graphene nanosheets (BGNs) with 2.2 atomic percentage (at%) boron doping level and a hierarchical microstructure were synthesized by an atmospheric-pressure carbothermal reaction as a noble-metal free catalyst for sensing H2O2. The isolated boron atoms on the BGNs surface act as the electrocatalytic sites by transferring charges to neighbor carbon atoms, and the hierarchical microstructure provides multidimensional electron transport pathways for charge transfer and therefore enhances the electrocatalytic ability. BGNs possess a higher reduction current in the cyclic voltammetry (CV) measurement than that of pristine graphene nanosheets (GNs) over the detection range of 0.0 to 10.0 mM at −0.4 V (vs. Ag/AgCl). The BGNs modified electrochemical sensor shows a linear range from 1.0 to 20.0 mM of H2O2 with a sensitivity of 266.7 ± 3.8 μA mM−1 cm−2 and limit of detection (LOD) of 3.8 μM at a signal-to-noise (S/N) ratio of 3. The beneficial hierarchical microstructure and the synergetic effects arising from doping boron in GNs accomplish the better performance of the BGNs modified electrochemical sensor.