The Yellow River Basin, a pivotal concentration area for China's population and industry, suffers from acute water shortages, ecological decline, and environmental pollution. Investigating the region's human-nature interaction is crucial for enhancing ecological conservation and sustainable development. However, research on this relationship at a grid scale, essential for national spatial planning and ecological management, remains insufficient. In this study, we utilized the Human Footprint (HF) and the Ecosystem Quality Index (EQI) as indicators to represent the human-nature interface. Employing the Sen+MK trend analysis, we delineated the spatio-temporal dynamics of human activities and ecosystem quality across the basin at a 1km×1km grid scale from 2000 to 2020. The four-quadrant diagram was applied to quantify the synergies and conflicts between the two. Moreover, various regression models were used to evaluate the degree and spatio-temporal variability of human impacts on ecosystem quality within the basin. The study revealed that both the EQI and HF in the Yellow River Basin exhibited an overall upward trend from 2000 to 2020. Areas with significant increases in EQI accounted for 64.2% of the region, while those for HF comprised 49.2%. The basin exhibits a trend of coordinated human-nature evolution. Coordinated interactions predominantly occur on the Loess Plateau, the western Taihang foothills, and along Ningxia's Yellow River areas. Conflict zones, constituting only 8%, are mainly in rapidly urbanizing regions and upstream areas with delicate ecosystems. Controlling for natural variables like temperature, precipitation, sunlight, and elevation, HF significantly and negatively impacts the EQI, with each 1% increase in HF decreasing EQI by 0.067%. This effect exhibits spatio-temporal heterogeneity, being more pronounced in the middle and lower Yellow River Basin. The overall negative influence of HF on EQI is gradually intensifying. The study introduces a novel methodology for grid-scale human-nature interaction research and informs policy development for human-nature harmony in the Yellow River Basin.