The hydrogen bonding interactions are potentially employed to fabricate high-performance room-temperature gas sensors for detection of organophosphorus. Nevertheless, the low response toward target gases of conventional sensing materials hinders their further applications. Herein, an isolated Cu-N5 sites engineering strategy is reported to improve sensing performances for polypyrrole-reduced graphene oxide hybrids (PPy-rGO)-based room-temperature dimethyl methyl phosphate (DMMP) sensors. The isolated Cu-N5 sites were simply constructed by adsorption of Cu2+ ions with PPy-rGO hybrids owing to strong metal-support interactions. X-ray absorption fine structure analysis indicates that Cux+ ions with mean chemical valence of + 1.07 nearly atomically distributed on support with coordination structure of Cu-N5. Benefiting from the structure regulation by Cux+ ions, as-constructed DMMP sensor demonstrated 4.5-fold improvement in response to 100 ppm DMMP and 2.5-fold improvement in limit of detection, compared to PPy-rGO hybrids. The density functional theory, spectroscopic characterizations and quartz crystal microbalance tests prove that the construction of Cu-N5 sites not only heightens hydrogen bonding interactions between NH bonds and DMMP molecules induced by newly formed coordination interactions between Cux+ ions and NH bonds, but also serves as new active sites for adsorption of DMMP molecules. This work offers a new avenue for developing high-performance room-temperature gas sensors by heightening hydrogen bonding interactions.