Abstract: Plant vital activities could be revealed on protein level. Obtaining good twodimensional gel electrophoresis(2DE) profile is the precondition for experimental procedures such as mass spectrum analysis, and so on. In order to explore the mechanism of mycorrhiza underlying drought resistance of Pinus massoniana on protein level, a set of optimized experiment system of 2DE for analyzing root protein extracted from P.massoniana has been established. The ideal 2DE profile could be obtained when protein was extracted by phenol extraction and shattered the cells with ultrasonic crasher, and then the gels were stained by fast silver stain and washed three times for 5 min after sensitizing and dying. The suitable concentration of protein was 300 μg per IPG gel. It also provided reference for the proteomic studies on other pine species.