Taking Sillago sihama Forsskål as the research object, through the optimization of the extraction method, hydration and isoelectric focusing, a two-dimensional electrophoresis system was established for spleen proteome of S. sihama Forsskål. The results showed that the twp-step hydration and isoelectric focusing method is better than the hydration and isoelectric focusing integrated method. The protein spots of the spleen of S. sihama Forsskål were detected by two-dimensional electrophoresis mainly at pH 4.0-7.0. The establishment of this technical system will lay a foundation for further research on proteomics of S. sihama Forsskål in the future.