A novel non-enzymatic glucose sensor has been constructed by using NiO hollow spheres (NiO-HSs) as sensing materials, which were prepared by a glycerin-assisted hydrothermal synthesis method. The analytical techniques including X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) indicate the successful formation of NiO-HSs, assembled by NiO nanoflakes with the length of about 500nm and the width of about 50nm. It was found that the resulting NiO-HSs exhibit good catalytic activity toward the oxidation of glucose in 0.1M NaOH, leading to a non-enzymatic glucose sensor with a fast amperometric response time of less than 3s, and the detection limit is estimated to be 0.3 μM at a signal-to-noise ratio of 3. Furthermore, this sensor shows good response to glucose in comparison to other normally co-existing electroactive species (such as dopamine, ascorbic acid and uric acid).