The NiO/Nitrogen doped carbon sphere (NiO/NCS) composites were successfully achieved via an easy one pot synthetic method with urea acting as both nitrogen source and Ni precipitator. The electrocatalytic performances of the obtained NiO/NCS modified glass carbon electrodes showed superior activity for direct electrocatalytic oxidation of glucose than that of nitrogen free NiO/carbon sphere (NiO/CS), which might be due to the synergistic effect of the properties of NCS and NiO nanoparticles. The introduce of nitrogen can improve the conductivity of the NiO/NCS and accordingly accelerate the electron transport within the composites, which was very beneficial to improve the sensitivity to glucose detection for NiO/NCS modified electrodes. The NiO/NCS electrodes exhibited two corresponding linear regions of 1–800 μM and 4–9 mM with the sensitivity of 398.57 μA mM−1 cm−2 and 17.81 μA mM−1 cm−2, and the detection limit of 0.25 μM and 0.05 mM respectively. Moreover, the NiO/NCS composites have also exhibited good selectivity by adding certain amount of urea, NaCl, l-proline, l-valine, l-Leucine and ascorbic acid into the 0.1 M NaOH solution, respectively. The high sensitivity, wide glucose detection range and good selectivity of the electrodes may ensure its potential applications in the clinical diagnosis of diabetes.