In this study, three precipitation protocols of protein extraction for 2-DE proteomic analysis were used to extract the protein from leaf tissue of four Rhododendron species, and the extraction efficiency, solubleness, integrity of protein extractions were compared. The results indicated that the TCA-acetone protocol for protein extraction cannot be used to the Rhododendron.L because the extracted proteins were ropy and difficult to be redissolved. Though extracted proteins with phenol protocol were eutectic and the spots in the 2-DE maps were abundant, however the impurities were too much and the method is inapplicable to R. simsii. The TCA-acetone-phenol protocol displayed the best result with spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility，and is applicable to four Rhododendron species. So TCA-acetone-phenol protocol is considered the best protocol of the protein extraction for 2-DE proteomic analysis of Rhododendron leaves.