Improving the efficiency of glucose oxidation reaction (GOR) is extremely important to build high performance nonenzymatic glucose sensors and fuel cells. In this work, we designed a novel binary (α–β) NiS/PANI nanorods electrocatalyst with polyaniline (PANI) as the substrates and binary (α–β) NiS nanoparticles dispersing on the PANI nanorods. The as-prepared NiS/PANI nanorods were characterized by XRD, XPS and SEM. The electrochemical performance of NiS/PANI nanorods was evaluated by cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy, which showed highly improved catalytic efficiency for GOR. When used as anodic catalysts in nonenzymatic glucose fuel cells, NiS/PANI nanorods displayed much higher power density of 1343.39 μW⋅cm−2 with an open circuit voltage of 0.84 V. NiS/PANI/NiS nanorods also presented excellent nonenzymatic sensing performance for glucose detection including a wide sensitivity of 682.21 μA⋅mM−1⋅cm−2 (10–9000 μM) and the low detection limit of 3.33 μM (S/N=3). The obviously improved electrochemical properties of NiS/PANI/NiS nanorods for GOR may be due to the synergistic effect of binary (α–β) NiS and PANI nanorods.