Desert oases are vital for maintaining the ecological balance in arid regions’ inland river basins. However, fine-grained assessments of water stress in desert oasis ecosystems are limited. In our study, we aimed to evaluate the water stress in desert oasis ecosystems in the middle reaches of the Keriya River Basin, with a specific focus on their ecological functions and optimizing water resource management. We hypothesized that evapotranspiration has significant effects on ecological water consumption. First, we estimated the actual evapotranspiration (ET) and potential evapotranspiration (PET) based on the SEBS (surface energy balance system) model and remote sensing downscaling model. Then, the ecological water requirement (EWR) and ecological water stress (EWS) index were constructed to evaluate the ecological water resource utilization. Finally, we explored the influencing factors and proposed coping strategies. It was found that regions with higher ET values were mainly concentrated along the Keriya River and its adjacent farmland areas, while the lower values were observed in bare land or grassland areas. The total EWR exhibited the sequence of grassland > cropland > forest, while the EWR per unit area followed the opposite order. The grassland’s EWS showed a distinct seasonal response, with severe, moderate, and mild water shortages and water plenitude corresponding to spring, summer, autumn, and winter, respectively. In contrast, the land use types with the lowest EWS were water areas that remained in a state of water plentitude grade (0.08–0.20) throughout the year. Temperature and vegetation index were identified as the primary influencing factors. Overall, this study provides a reliable method for evaluating the EWR and EWS values of basin scale vegetation, which can serve as a scientific basis for formulating water resource management and regulation policies in the region.