Pt-Ni/MWCNTs composite catalyst was successfully synthesized with platinum and nickel supported on multi-walled carbon nanotubes (MWCNTs) by impregnation-reduction method. The Pt-Ni/MWCNTs catalyst was characterized by transmission electron microscopy (TEM) and X–ray powder diffraction (XRD). The results show that Pt and Ni were uniformly loaded onto the carbon nanotubes. The Pt-Ni composite catalyst has a approximate particle size of 2 nm. XRD demonstrate that the composite catalyst is a solid solution structure based on Pt crystal lattice. The electrochemical performance of the modified electrode was evaluated by cyclic voltammetry and chronoamperometry in alkaline media. Electrochemical measurements indicate Pt-Ni/MWCNTs could directly catalyze the oxidation of glucose and a selectivity of 571.68 μA•mmol/(L•cm2) towards glucose oxidation was obtained. The composite catalyst displayed a wide linear range from 10 μmol/L to 8.2 mmol/L (R=0.9994) with the limit of detection of 0.5 μmol/L (S/N=3). The sensor exhibits excellent selectivity, stability and repeatability. The interferences from the oxidation of ascorbic acid, uric acid and dopamine could be effectively avoided.