This paper takes a systematic study on the experimental conditions using the grinding method and stepping dissolution technique to extract the fluid phase and sulfide phase from sphalerite for Rb-Sr isochrone dating, and the entire analysis process was established and improved. Through the demonstration of Rb-Sr dating method used in Dagoudong Pb-Zn deposits, western Hunan Province, the validity of the method was further verified and showed a good application prospects. The isochrone ages indicated that Dagoudong deposit was formed in Early Ordovician, and maybe related to later diagenesis or regional tectono-thermal events.