3D nanoflower-like CuxO/multilayer graphene composites (CuMGCs) have been successfully synthesized as a new type of room temperature NOx 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 CuxO 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 NOx, the CuMGCs achieved a higher response at room temperature than that of the corresponding CuxO. In detail, the CuMGCs show a higher NOx 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 NOx 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 CuxO and can easily capture and migrate electrons from the conduction band, resulting in the enhancement of electrical conductivity.