3D nanoflower-like Cu_xO/multilayer graphene composites (CuMGCs) have been successfully synthesized as a new type of room temperature NO_x gas sensor. Firstly, the expanded graphite (EG) was activated by KOH and many moderate functional groups were generated; secondly, Cu(CH₃COO)₂ and CTAB underwent full infusion into the interlayers of activated EG (aEG) by means of a vacuum-assisted technique and then reacted with the functional groups of aEG accompanied by the exfoliation of aEG via reflux. Eventually, the 3D nanoflower consisting of 5–9 nm Cu_xO nanoparticles homogeneously grow in situ on aEG. The KOH activation of EG plays a key role in the uniform formation of CuMGCs. When being used as gas sensors for detection of NO_x, the CuMGCs achieved a higher response at room temperature than that of the corresponding Cu_xO. In detail, the CuMGCs show a higher NO_x gas sensing performance with low detection limit of 97 ppb, high gas response of 95.1% and short response time of 9.6 s to 97.0 ppm NO_x at room temperature. Meanwhile, the CuMGC sensor presents a favorable linearity, good selectivity and stability. The enhancement of the sensing response is mainly attributed to the improved conductivity of the CuMGCs. A series of Mott–Schottky and EIS measurements demonstrated that the CuMGCs have much higher donor densities than Cu_xO and can easily capture and migrate electrons from the conduction band, resulting in the enhancement of electrical conductivity.