Pt-Ni alloy nanoparticles with different atomic ratios of Pt to Ni (1:3；1:1；3:1) were prepared by colloidal synthesis method with Pt(acac)2 and Ni(ac)2?4H2O as precursors, respectively, and Pt-Ni/C bi-metallic catalysts were obtained after the nanoparticles were loaded on the surface of XC-72 carbon supports, and Pt3Ni/C catalysts displayed the best catalytic activity. The peak current density of Pt3Ni/C catalyst was 42.5 mA.cm-2 at the positive scanning, which was 3.2, 5.3 and 1.2 times as high as those of PtNi/C, PtNi3/C and Pt/C catalysts, and else the anti-poisoning ability of PtNi3/C was the strongest (If/Ib was 23.5) contorted with other samplers. TEM and XRD analysis indicated that Pt-Ni bi-metallic nanoparticles are uniformly distributed with the sizes ranging from 2 to 4 nm, and XPS demonstrated that the addition of Ni changes the external electronic structure of Pt, reduces the absorption of CO on Pt and releases more Pt active sites, which was the reason for the electrocatalytic activity and anti-poisoning ability of Pt-Ni/C catalyst improved.