Nile tilapia (Oreochromis niloticus) is a tropical species that prefers to live in shallow waters, and it has the merits of fast growth and high reprodu-ctive rate, also has the characteristics of less bone spurs, thick fleshy, which has been widely cultured in southern China, and China is by far the largest producer of farmed Nile tilapia. In order to establish a two-dimensional electrophoresis (2-DE) system for proteomic analysis of Nile tilapia muscle, protein samples of Nile tilapia muscle were separated using 2-DE after extracting. By optimizing different preparation methods, different pH of immobilized pH gradients(IPG) gel strips and their lengths, as well as different concentration of SDS-PAGE gel and staining methods (silver staining and Coomassie blue staining), we obtained the best 2-DE experiment conditions. The results show that the optimum method for protein samples preparation of Nile tilapia muscle is combination of liquid nitrogen grinding and acetone precipitation. For 2-DE assay, using 24 cm pH 4-7 IPG gel strips for isoelectric focusing(IEF) and 12.5% gel for the second SDS-PAGE. Besides, dyeing the gels by silver staining could get the optimized 2-DE map of Nile tilapia muscle protein with excellent degree of separation, clear protein points, high resolution and less cross band. The 2-DE technical system was successfully established in this study, which could be used for the separation of proteins in the future Nile tilapia muscle proteomics research.