To study proteome of Rhizoctonia solani AG1 IA, the causal agent of rice sheath blight disease, three protein extraction methods (TCA-acetone precipitation method, SDS method and TCA-acetone-phenol/SDS method) were compared and a two-dimensional electrophoresis procedure was set up. Gel electrophoresis patterns were analyzed by Quantity One software and the results showed that 557, 309 and 877 protein spots were calculated by using TCA-acetone precipitation method, SDS method and TCA-acetonephenol/SDS method, respectively. Protein extracted by SDS method contained impurities such as salt ions and small molecules, and exhibited the fewest protein spots. Protein obtained by TCA-acetone precipitation method was yellow, indicating pigments contamination. Finally, a combined TCA-acetone precipitation and SDS method could get rid of salt ions, small molecules and pigments and produce a clear and high resolution electrophoresis pattern. Therefore, an optimized TCA-acetone-phenol/SDS extraction method was established in this study, which was suitable for proteins extraction from R. solani AG-1 IA for two-dimensional electrophoresis.