The rich classes of secondary metabolites from the genus Sorangium have been an important source of new drugs. The proteome analysis is an effective method to study the regulation of metabolism. However, the genus Sorangium contains a large amount of exopolysaccharides or slime that interferes with protein solubility, resolution, and repeatability in proteome analysis. To perform high-throughput screening of the specific proteins expressed by Sorangium cellulosum So0157-2, we optimized the two-dimensional electrophoresis (2-DE) protocol. Firstly, the proteins have better solubility in lysis buffer. The pH 3～10 NL strip is appropriate for the first-dimensional isoelectric focusing, improving the resolution of protein spots. 1 mg of protein was used in the isoelectric focusing, improving the expression of low-accumulation proteins. 15% SDS-PAGE improved the resolution and repeatability for separation of these proteins. Based on the optimal 2-DE protocol, the protein patterns of S. cellulosum So0157-2 cultured in M26 medium for three days were acquired, and 552 protein spots were detected. Further, the expressed proteins (85.9%) were identified by MALDI-TOF-MS. The identified proteins included components of cell structure and function, and cell metabolic enzymes. Worthy to be mentioned, 8 proteins were related to the transformation and metabolism of carbohydrate, which were contributed to the in-depth study of epothiloneoside A. This optimal protocol laid the foundation for the further construction of proteome expression database of S. cellulosum So0157-2 in various industrial culture conditions.