To develop an efficient and high resolution 2-DE procedure for wheat root proteome analysis, the method of wheat root protein extraction and sample preparation for two-dimensional gel electrophoresis （2-DE） were optimized by using the roots of wheat cultivars Jing-411 and Chinese Spring at two leaves stage. Separation results of wheat root protein by using the optimized method showed that better resolution of the electrophoresis patterns could be achieved. In particular, the protein samples extracted from Trizol reagent appeared to be better for two-dimensional gel electrophoresis separation, which could be used as an important tool for the studies on the wheat root proteome.