In this study, we reported the fabrication and characterization of CuO nanospheres based nonenzymatic glucose sensor. CuO nanospheres, consisting of many small and uniform nanoneedles with tips about tens of nanometers in diameter, were first synthesized by a simple, convenient, template-free, and one-step hydrothermal method, and then facilely co-deposited on a glassy carbon (GC) electrode with Nafion. The CuO modified GC (Nafion/CuO/GC) electrode shows excellent oxidation of glucose in basic solution and high sensitivity to glucose concentration change. Moreover, the presence of a high concentration of chloride ions does not affect the sensor's performance. These results demonstrate CuO nanospheres possess excellent electrocatalytic properties and are attractive in electrochemical detection.