The Yellow River Estuary as a vital ecological bridge linking freshwater and marine ecosystems, with its ecological health significantly impacted by the Yellow River water and sediment regulation project. To assess the environmental ecological quality of the estuary during this regulation period, data on macrobenthos and bottom water environmental factors were collected on three occasions throughout the 2023 water and sediment regulation phase. Four indices—AMBI, M-AMBI, BOPA, and Shannon-Wiener diversity—were employed to gauge the ecological status of the estuary. To reconcile the discrepancies in evaluation standards among these biological indices, an endeavor was made to establish a comprehensive biological index (CBI) using the entropy weight method. This approach aimed to unify the evaluation criteria across the four indices. Furthermore, the Nemerow Comprehensive Water Quality Evaluation Index (P) was introduced to validate the results. The survey revealed a total of 133 macrobenthos species belonging to five taxa, with polychaetes and mollusks emerging as the dominant groups. Notable shifts were observed in the macrobenthos community structure across the three periods: before, during, and after the water and sediment regulation. During the regulation period, the benthic ecological quality of the Yellow River Estuary was generally classified as “moderate” or “mildly polluted”. Pollution levels intensified at stations nearer to the estuary, indicating poorer ecological quality in these areas. The CBI exhibited a strong correlation with bottom water environmental factors and aligned well with the findings of the comprehensive water quality evaluation index. The evaluation grades for the ecological status at each monitoring station were distinctly stratified, demonstrating that the CBI method, constructed using the entropy weight method for various biological indices, is a viable tool for assessing the environmental ecological quality of the Yellow River Estuary. These research findings offer fresh perspectives for evaluating the environmental ecological quality of this crucial estuary.