The effect of Au and/or Ni addition on the ethanol oxidation reaction (EOR) performance in alkaline media of Pd-based binary and ternary catalysts (Pd 3Au/C, Pd3Ni/C, and Pd3AuNi/C) is systematically elucidated. The EOR activities, structures, morphologies, surface compositions and surface species of the prepared catalysts are analyzed by cyclic voltammetry, X-ray diffraction and X-ray absorption spectroscopy, high resolution transmission electron microscopy, X-ray photoelectron spectroscopy, and temperature-programmed reduction, respectively. It is observed that the surface Ni with the chemical state of NiOOH can promote the EOR through bi-functional mechanism and spillover while surface Au can modify the Pd lattice and electron configuration which is helpful for the absorption of ethanol molecular. Chronoamperometric (CA) results obtained at room temperature demonstrate that the mass current density of ternary Pd3AuNi/C catalysts after the long-term EOR test for 4 h is about 1.39 and 1.10 times higher than that of the monometallic Pd/C and binary Pd3Au/C catalysts, respectively. It is proposed that the EOR stability enhancement of Pd3AuNi can be attributed to the synergistic effect of Ni and Au alloying.