Abstract: Within the framework of traditional translatory flow, infiltrating precipitation and water at any soil depth is firstly well mixed and eventually enters the stream. Based on the dual stable isotope approach (δD and δ¹⁸O), recent studies showed that ecohydrologic separation occurs during the mixing process between precipitation and soil water. Namely, soil water has two pools: soil bound water which includes unavailable and available water used by plants, and soil mobile water entering the stream. The partial mixing of both water pools is defined as hydrologic connectivity. In this review, the concept and meaning of ecohydrologic separation are explained systematically. We described the mixing process between precipitation and soil water, and water isotopes (δD and δ¹⁸O) of soil bound water and mobile water in detail. We summarized the advantages and disadvantages of the direct and substitute methods to measure δD and δ¹⁸O in soil water, bound water, and mobile water. We reviewed the researches on hydrologic separation and connectivity of soil bound water and mobile water in runoff plot and watershed, including the qualitative research based on the direct and substitute methods, and the quantitative research using the models and control experiments. At last, we proposed that further studies should strengthen the research on the qualitative and quantitative methods of ecohydrological separation, and their influences on traditional ecohydrology models.