Abstract: The aim was to establish an efficient Two-Dimensional Electrophoresis (2-DE) ptotocol for proteomic method of livers from muscovy ducks which were experimentally infected with Norvel Duck Reovirus (NDRV). The conditions which included the reagent of Lysis buffer, amount of loaded protein and parameters of Isoelectric Focusing (IEF) were compared and optimized. The pathological changes in livers were simultaneously observed. The results showed that the livers with characteristic pathology were chosen to be the samples, which were from muscovy ducks at the fifth day after inoculated with NDRV. The high-resolution and good-repeatability 2-DE images were obtained with 1100 μg of liver protein samples which were extracted by Lysis-buffer, purified by 2D clean-up kit, separated by 2-DE under suitable electrophoresis parameters, and stained with colloidal Coomassie brilliant blue G-250 solution. Analyzed with the software PDQest 8.0, 26 differentially expressed proteins spots were detected between the NDRV-NP03 infected group and the mock-infected group, of which the expression levels were more than 3 times in expression level. The established 2-DE method would pave the way for the further study on the host cell protein and differential proteomic analysis of waterfowl reovirus.