Thoroughly investigating the evolution of groundwater circulation and its controlling mechanism in the Aksu River Basin, where human activities are intensifying and the groundwater environment is increasingly deteriorating, is highly urgent and important for promoting the theory, development and implementation of groundwater flow systems (GFSs) and protecting groundwater resources. Based on a detailed analysis of the sediment grain size distribution, chronology, electrofacies, glacial sedimentary sequence, palaeoclimate indicators and existing groundwater age, this paper systematically reconstructs the palaeosedimentary environment of the basin-scale aquifer system in the study area and scientifically reveals the evolutionary pattern and formation mechanism of the GFS. The results showed that the later period of the late Pleistocene experienced a rapid downcutting erosional event caused by tectonic uplift, and the sedimentary environment transitioned from a dry–cold deep downcutting environment in the Last Glacial Maximum (LGM) to a coarse-grained fast-filling fluvial facies sedimentary environment in the Last Glacial Deglaciation (LDP) as the temperature rose; then, it shifted to an environment of fine-grained stable alternating accumulation of fluvial facies and lacustrine facies that was dominated by the warm and arid conditions of the Holocene megathermal period (HMP); this process changed the previous river base level via erosion, glacier elongation or shortening and river level, thus resulting in a complex coupling relationship between the palaeosedimentary environment, palaeoclimate and basin GFS. Furthermore, the existing GFS pattern in the basin exhibits a vertically unconformable groundwater age distribution, which indicates that it is the outcome of the complex superposition of groundwater flow controlled by the palaeosedimentary environment in different periods. Therefore, neotectonic movement and climate fluctuation have jointly acted on the variation in the river level, resulting in the “seesaw” effect, thereby fundamentally controlling the strength of the driving force of groundwater and resulting in the gradual evolution of the GFS from the fully developed regional GFS pattern during the LGM to the current multihierarchy nested GFS pattern.