[Objective] The water use characteristics of plants and their responses to environmental changes in a tunnel construction area at the eastern margin of Qinghai-Xizang Plateau were studied in order to provide a reference for soil and water conservation in this ecologically fragile region influenced by both nature and human activity. [Methods] The δ²H and δ¹⁸O values of xylem water of typical plants and potential water sources in different months from 2020 to 2021 in a tunnel construction area with an altitude of 3 230 m were measured. The MixSIAR model was used to calculate the proportion of plants absorbing water from each potential water source. The proportional similarity index (PS index) between every two plants was calculated to compare the water competition relationship between different species. [Results] Shallow soil water (0—40 cm) was the main water source for Festuca rubra, Picea likiangensis var. balfouriana, and Quercus spinosa. Populus szechuanica mainly absorbed middle and deep (40—200 cm) soil water. The uptake proportions from shallow soil water by plants in August and October 2021 were significantly lower than during the same period in 2020. For P. likiangensis var. balfouriana, there were significant positive and negative correlations (p＜0.05) between soil water utilization ratio and soil water content in the 0—40 cm and 40—80 cm layers, respectively. The water competition relationships between P. szechuanica and the other three plants in October 2020 were weak, with the PS index ranging from 0.19 to 0.54. [Conclusion] Changes in soil moisture content and external temperature were the main factors influencing the variation of vegetation water use strategy at the eastern margin of the Qinghai-Xizang Plateau, and the importance of these two factors varied among different species. Loss of soil and soil water due to tunnel construction resulted in a decrease in soil water content after tunnel construction. The contribution of shallow soil water to plants was lower after tunnel construction than before tunnel construction. Therefore, the impact of tunnel construction on soil water and vegetation adaptation mechanisms in ecologically fragile areas needs to be of concern.