Understanding the isotopic composition and interrelations of different water bodies at the soil–plant–atmosphere continuum (SPAC) is crucial to reveal the processes and mechanisms of regional water cycles. Rainfall, river water, plant, and soil samples from Lanzhou City, China, were collected from April to October 2016. The hydrogen (δ2H) and oxygen (δ18O) of the local precipitation, river water, soil water, plant xylem water, and leaf water were determined. We found that trees mainly uptake the middle (30–60 cm) and deep (60–100 cm) layer soil water during the growing season, and the shrubs mainly uptake the middle soil water. All herbs uptake the shallow soil water (0–30 cm) during the growing season. The δ18O of shallow soil water was found to be isotopic-enriched because of evaporation and exhibited a decline from the shallow soil layer towards the deeper layer. The variation of δ18O and soil water content (SWC) was remarkable in shallow soil, which was mainly due to evaporation and precipitation infiltration, while water in the middle and deep layer was less affected by these phenomena.