Abstract: This paper used the method of TCA/acetone to extract all proteins of phloem in tetraploid Robinia pseudoacacia. A twodimensionalelectrophoresis system for proteomic analysis of tetraploid Robinia pseudoacacia was established by optimizing the parametersincluding the pH gradient of IPG strip, the gel concentration, the loading quantity of sample and isoelectric focusing conditions. The resultsshowed that ：Use the TCA/acetone method to extract all phloem protein of tetraploid Robinia pseudoacacia, choose 17 cm IPG strip withpH4-7, load protein samples of 550 μg, followed staining with colloidal Coomassie Brilliant Blue and use 12% SDS-PAGE for tetraploid Robiniapseudoacacia phloem total proteins in two-dimensional electrophoresis while isoelectric focusing IEF hours increased from 60 000 Vh to 80 000Vh can get a clear background with more protein points, high resolution and good repeatability. Using the established system of two-dimensionalelectrophoresis can dug protein points from the gel directly and send them to mass spectrometry. Use this system to analysis differences ofhardwood cuttings rooting callus phase protein expression in tetraploid Robinia pseudoacacia. Filter out 83 differential protein spots, amongwhich, 15 were higher, 22 were novel, 22 were lower and 24 were lack of expression.