N-doped carbon support can increase nanoparticle (NPs)-support interaction and generate more defective sites to promote prepared catalyst's performance. Adenine can provide a large amount of nitrogen resources and possesses great potential in preparing N-doped carbon support. Herein, in this study, we prepare a catalyst through supporting Pd3Ru nanoparticles on an adenine-originated N-doped carbon support. Electrochemical results indicate that the adenine-originated N-doped carbon supporting on Pd3Ru NPs (Pd3Ru/NC) shows high catalytic performance in comparison with Pd/NC and Pd/C in glycerol electrooxidation. Furthermore, the activity, poisoning tolerance, long durability and stability of the Pd3Ru/NC catalyst are greatly promoted. These results may be brought about by defective sites of adenine-originated NC favoring good NPs dispersion and nucleation, and the bifunctional effects of Pd3Ru alloy, as well as the good interaction between NPs and NC support. All results clearly demonstrate that as-prepared adenine-originated NC support can greatly improve catalytic performance.