The 3D NiO hollow sphere/reduced graphene oxide (rGO) composite was synthesized according to the coordinating etching and precipitating process by using Cu 2 O nanosphere/graphene oxide (GO) composite as template. The morphology, structure, and composition of the materials were characterized by SEM, TEM, HRTEM, XPS, and Raman spectra, and the electrochemical properties were studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry. Moreover, the electrochemical activity of the composite materials with different morphologies were also investigated, which indicating a better combination of the NiO hollow sphere and the rGO. Used as glucose sensing material, the 3D NiO hollow sphere/rGO composite modified electrode exhibits high sensitivity of ~2.04 mA mM −1 cm −2 , quick response time of less than 5 s, good stability, selectivity, and reproducibility. Its application for the detection of glucose in human blood serum sample shows acceptable recovery and R.S.D. values. The outstanding glucose sensing performance should be attributed to the unique 3D hierarchical porous superstructure of the composite, especially for its enhanced electron-transfer kinetic properties.Electrochemical biosensors have been extensively applied to detect biological substances via catalysis and recognition behaviors happening on the surface of electrodes in the fields of medicine, food, industry and environment 1-4 . The generation of electrochemical signal normally includes electrocatalytic reaction happening at the electrolyte/electrode interface, and the electron transfer inside the electrode 5,6 . Intimate correlation of sensing performance and the structural and electrocatalytic properties of electrodes has motivated great efforts to the design of new materials with superior electrocatalytic activity and electron-transfer kinetics to achieve rapid and sensitive response of electrochemical signal in biosensor 7,8 .Metal oxides play an important role in the miniaturization of glucose biosensor due to their inexpensive, good biocompatibility, and excellent electrocatalytic activity along with the controllability of the structure and morphology 9-11 . The effective application of metal oxides is prospective to break through the pivotal limitations of the costly enzymes since the typical glucose oxidase is intrinsically susceptible to the physical and chemical environments 1, 12-14 . Nanostructured metal oxides, such as zero-dimensional (0D) particles, 1D nanowires, 2D nanosheets, and some hollow structures have been widely studied as electrode materials for glucose biosensors with improved sensitivity, reproducibility, and stability. Nickel-based materials, such as NiO and Ni(OH) 2 have been extensively research as electrocatalyst for glucose due to its redox couple of Ni 3+ /Ni 2+ in the alkaline medium. However, the poor electronic conductivity of nickel-based materials at room temperature determines the inferior electron-transfer kinetics of the constructed electrodes, which significantly hinders ...