The ratio of actual to potential evapotranspiration (AET/PET) represents the water supply of terrestrial ecosystems and can be used to assess the water stress level of plants or ecosystems. This study quantified the control of meteorological, hydrological, and biological factors on AET/PET variations using variance partitioning analysis and path analysis. A soil–plant–atmosphere continuum model was validated and used to simulate AET and PET, as well as the interannual dynamics of AET/PET in typical ecosystems in the upper, middle, and downstream reaches of the Heihe River Basin from 2014 to 2016. The good agreement between measurements and simulations indicates that the model accurately represents the water/heat transfer process in various ecosystems within the Heihe River Basin. The study found that the alpine meadow ecosystem had the highest annual average AET/PET (0.89 ± 0.12), followed by the Populus euphratica ecosystem (0.72 ± 0.17) and cropland ecosystem (0.65 ± 0.15). AET/PET variation characteristics were comparable across the three ecosystems, showing apparent seasonality and interannual fluctuations. Plant transpiration stress was found to be a dominant indicator of ecosystem water availability. Changes in AET/PET were attributed to diverse environmental conditions in the three distinct ecosystems. While the relationship between soil water (SW) content and AET/PET weakened in cropland ecosystems with low water stress, the study demonstrates that SW content remains the most important factor governing AET/PET. The leaf are index and vapour pressure deficiency had significant indirect effects on AET/PET by influencing SW content. Overall, this study provides scientific support for optimizing water resource management in the Heihe River Basin by revealing the interannual variations and controlling factors of AET/PET in typical ecosystems.