The major hindrance for a successful commercialization of direct ethanol fuel cells (DEFCs) is the sluggish ethanol oxidation reaction (EOR) and the rapid poisoning of anodic catalysts by by-products. In this work, a high-performance PdNi nanowire (PdNi-NWs) catalyst was prepared via a two-step process in polyol medium based on the galvanic replacement reaction. By carefully screening parameters for a uniform deposition of Pd layers on Ni nanowires, the PdNi-NWs electrode exhibits a significant enhancement of both catalytic activity and durability for the EOR in alkaline media, which may be ascribed to the electronic structure modification and bifunctional electrocatalytic mechanism with hydroxyl and ethanol bindings on Ni and Pd, respectively. Moreover, these nanowire structures are efficient electron and mass transfer and enriched with abundant active sites by oxygen-rich compounds of Ni. The highest electrocatalytic performance has achieved with a low Pd:Ni molar ratio of 18:100, which reaches 9.3 times superior mass activity and the EOR onset potential shifts 50 mV negatively compared with Pd nanoparticles (PdNPs). These results highlight the active role of PdNi-NWs catalysts in DEFCs.