Abstract: To establish a method of twodimensional electrophoresis (2DE) in good repetition and high resolution for proteomic analysis of bursa of Fabricius, the bursa of Fabricius from specific pathogen free chickens at different days old were taken to analyses by 2DE. The firstdimensional electrophoresis was immobilized pH gradients (IPG) strips isoelectric focusing and seconddimensional electrophoresis was vertical electrophoresis of SDSPAGE. The conditions for sample preparation, rehydration, isoelectric focusing, equilibration, staining and other steps were modified. The results were analyzed with the software PDQuest801. More than 800 spots on 17 cm IPG strip (pH 58) for normal bursa of Fabricius tissue were detected with this method, and the match rate of protein spots was 835%. Comparative analysis of bursa of Fabricius at different ages in multiple 2DE gels revealed 37 differentially expressed protein spots, including 17 upregulated protein spots，11 downregulated protein spots，5 new emerging protein spots and 4 disappear protein spots. The method of 2DE for proteomic of bursa of Fabricius has been established. It would provide a basis for further elucidation of development and immune function of bursa of Fabricius.