Focusing on sustainability of water resources and ecology in the complex karst critical zone, we illustrated functions of the hydro-geochemical analysis on hydrology from the aspects of connection and interaction among hydrology–vegetation–soils/rock fractures along the karst subsurface profile. We reviewed isotopic and geochemical interpretations on tracing water sources for plant uptake, quantifying watershed outlet flow composition and residence times, and evaluating long-term evolution among climate–landscape–hydrology in the karst critical zone. In this paper, the application of the hydro-geochemical analysis on the above aspects in the karst areas of southwest China was summarized. Focusing on sustainability of water resources and ecology in the complex karst critical zone, we illustrated functions of the hydro-geochemical analysis on hydrology from the aspects of connection and interaction among hydrology-vegetation-soils / rock fractures along the karst subsurface profile. isotopic and geochemical interpretations on tracing water sources for plant uptake, quantifying watershed outlet flow composition and residence times, and evaluating long-term evolution among climate-landscape-hydrology in the karst critical zone. In this paper, the application of the hydro-geochemical analysis on the above aspects in the karst areas of southwest China was summarized.