Ecohydrological regionalization formed the basis for water resources management and ecological protection, and exploring the evolution of ecohydrological processes under environment changes held great significance for water security and ecological security. Based on long-term multi-element, multi-scale, and multi-source datasets from the water conservation area of the Yellow River (1980—2019), this study constructed a comprehensive ecohydrological index system and divided primary and secondary ecohydrological regions. The analysis identified spatiotemporal evolution patterns and mutual feed-back effects among critical climate, ecological, and hydrological elements across different ecohydrological regions. The results showed that (1) the study area was divided into five primary and ten secondary ecohydrological regions based on the comprehensive ecohydrological index system while accounting for complex regional characteristics. (2) Temperature and leaf area index exhibited continuous increase during 1980—2019, while precipitation and snow depth showed overall declines during 1980—1999 but demonstrated increasing trends after 2000. (3) Rising temperature and precipitation promoted vegetation growth. A one-month lag effect of temperature on leaf area index was observed in areas with large elevation fluctuations, while in the source region of the Yellow River, the leaf area index-precipitation correlation peaked at a one-month lag. This study provided scientific references for ensuring water and ecological security in the Yellow River Basin.