Stable hydrogen and oxygen isotope analysis has been used widely to study the usage of plant water in ecosystems, especially in arid ecosystems. The theoretical basis of this analysis is that hydrogen and oxygen isotopes do not fractionate during the processes of root water absorption and water transport to the plant xylem. Dramatic differences in hydrogen and oxygen isotope ratios exist among various water sources within an ecosystem, which is a pre-condition of using stable hydrogen and oxygen isotope analysis to distinguish the proportional contributions of different water sources. However, some factors have caused uncertainty in the quantification of plant water sources using stable hydrogen and oxygen isotope analysis. This study has discussed previous research as examples of the factors leading to this uncertainty, such as the retention of root water uptake in relation to the dynamics of the isotopic ratio of water sources, the seasonal variation of isotopic water sources, and the variation of the isotopic ratio of water sources caused by evaporation and mixing of different water sources. Our research can act as a reference for future research into the quantification of plant water sources using stable hydrogen and oxygen isotopic analysis.