Abstract: The arid inland river basins in northwestern China have been experienced various stages of soil and water resources over-exploitation over the past 50 years, as well as ecological protection and restoration in recent years, which has significantly influenced the regional groundwater and ecological environment. However, the long-term evolution of groundwater and its ecological effects remain unclear. In this study, the Kongque River Basin is chosen as a study area, and the spatio-temporal evolution of groundwater flow field is analyzed based on the comparison of recent groundwater monitoring data and historical data. The ecological effects of long-term groundwater evolution are discussed by analyzing the influence of regional groundwater to Populus euphratica in the riparian zone. The results show that from 1971 to 2021, the groundwater flow in the Kongque River Basin has undergone drastic variation due to excessive groundwater exploitation, and the groundwater levels have shown a general downward trend. The total area where the groundwater levels have decreased by more than 40 m is 204.69 km², as well as 1735.81 and 1018.56 km² for 20−40 and 5−20 m, and eventually led to the formation of a regional drawdown funnel, which have seriously affected the water consumption of Populus euphratica in the middle and lower reaches. From 1971 to 2016, the regional groundwater level depth in the riparian zone of the Kongque River Basin decreased by more than 10 m, with a degradation and shrinkage of Populus euphratica. After the implementation of the ecological water transport project since 2016, the groundwater level depth has gradually increased by 2−4 m, and reached the critical groundwater level of Populus euphratica. From 2014 to 2018, the regional NDVI increased by 17%, which indicates that the coverage rate of natural vegetation has improved. This study can improve the understanding of evolution patterns in groundwater and ecological environment in arid inland river basins of the northwestern China, and provide scientific references for sustainable development and utilization of groundwater and ecological protection in similar regions.