In this paper, we successfully prepared Ru-doped Co3O4 flower-like hollow microspheres by a simple hydrothermal method. The microstructure characterization results indicated the formation of well-organized microflowers with a size of 0.5–1 µm, which were composed of nanosheets. The gas sensing performances of sensors by using the synthesized Ru-doped Co3O4 hollow microflowers as sensing material were investigated. The 1 at% Rudoped Co3O4 sensor showed the best acetone sensing performance and had a response 18.8–10 ppm acetone, which was almost 5.4 times that of pure Co3O4. Significantly, the sensor had an ultra-low detection limit, with a response value of 1.5–50 ppb acetone at 137.5 ◦C (30%RH). We propose a plausible mechanism for sensing performance improvement. Namely, the substitution of Ru4+ regulated the carrier concentration and induced the change of Co3O4 defect oxygen and chemisorbed oxygen.