Zinc oxide hexagonal prisms (ZnO) decorated with nickel nanoparticles (Ni) onto a glassy carbon by a facile two-step electrochemical strategy. The resulting Ni-ZnO modified electrode was characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and electrochemical methods. Incorporating the lager surface area of ZnO 3D scaffold and high electrocatalytic activity of Ni, the prepared Ni-ZnO modified electrode possessed strong electrocatalytic ability toward glucose. At the optimum conditions, the calibration curve for glucose determination was linear in the range of 1μM to 8.1mM, a high sensitivity of 824.34μAmM−1cm−2 and a low detection limit (S/N=3) of 0.28μM were obtained. In addition, the proposed sensor was successfully applied to analyze glucose level in human blood serum samples.