Semi-arid regions, such as the Yellow River Basin in China, are highly sensitive to environmental changes due to their scarce water resources and fragile ecosystems. This study explores the response mechanisms of eukaryotic plankton community structure to environmental conditions in the Dahei River Basin, a representative semi-arid region in central-western Inner Mongolia, across different seasons (spring, summer, autumn) and regions (hills, plain, urban). Our results reveal significant temporal and spatial variability in water resources and quality factors, which in turn affect eukaryotic plankton community structure. Using distance decay relationship (DDR) modeling and random forest analysis, we demonstrate that water quality factors, especially water temperature, are the primary factors influencing community composition in urban regions, where human activities such as industrial thermal discharges and urban heat island effects elevate water temperatures. In summer, increased water volume and flow velocity enhanced river connectivity, promoting greater community structure similarity across the basin, while in autumn, reduced flow and connectivity led to spatial heterogeneity and increased growth of the pollutant-tolerant species like Desmodesmus and Stephanocyclus. The synergistic effects of low flow rates and deteriorating water quality during the dry period intensified the influence of environmental factors on phytoplankton, decreasing community stability and increasing deterministic processes in community assembly. This shift towards determinism, especially in urban regions heavily impacted by human activities, highlights the sensitivity of semi-arid aquatic ecosystems. Co-occurrence network analysis further reveals that urban regions exhibit lower network complexity and stability, underscoring the heightened ecological sensitivity in these regions. In conclusion, the study highlights the critical role of water volume and quality in shaping eukaryotic plankton communities and elucidates how environmental changes, exacerbated by human activities and climate change, disrupt ecosystem stability. These findings offer essential insights for managing and conserving aquatic ecosystems in semi-arid regions facing dual pressures of climate change and human activity.