Abstract: To establish a twodimensional polyacrylamide gel electrophoresis (2D PAGE) system for leaf proteomic analysis of Hibiscus hamabo, three methods including lysisbuffer method, TCAacetone precipitation method, and the improved phenol extraction method were applied for leaf protein isolation and purification, and the key technologies of electrophoresis including the sample loading quantity, isoelectric focusing conditions, equilibrium time were optimized by taking 24 cm pH 310 immobilized pH gradient(IPG) strips. The results indicated that the reproducible 2D gel with more spots and high resolution electrophoresis diagram could be obtained by using the improved phenol extraction method, with 1000μg of loading protein sample per IPG strip, 90kV·h isoelectric focusing (IEF) time, 15 min of equilibrium time and staining with colloidal coomassie brilliant blue as the most suitable treatment conditions. The results of repetitive experiments showed that this electrophoresis system was stable and reproducible, which could meet the demand for the proteomics research on H. hamabo.