Abstract: As ecological indicator species, aquatic vascular plants and macrobenthos continuously reflect the ecological environment of rivers. To investigate the diversity, distribution characteristics, and their responses to environmental and socio-economic factors in the source section of the Yellow River main stream, we conducted field surveys across eight counties in the source region. Six biodiversity indices (density, biomass, species richness, Shannon diversity, Pielou's evenness, and Margalef's richness) of aquatic vascular plants and macrobenthos were calculated and analyzed. Using redundancy analysis (RDA) to determine influencing factors. The study recorded 6 aquatic vascular plant species and 27 macrobenthos species in the source section of the Yellow River main stream. In the study area, aquatic vascular plants exhibit significant spatial distribution variations. The biodiversity indices of aquatic vascular plants in the source section of the Yellow River main stream in Maduo County were the highest, while only Lysimachia maritima L. was collected in Gande County. No aquatic vascular plants were found growing in the other counties. Macrobenthos demonstrated a relatively wide and diverse distribution. Chlaenius sp. showed the broadest distribution range, Anisogammarus sp. reached the highest density, and Chironomidae represents the most abundant species. The highest density and species number of macrobenthos were in Dari County, while the highest biomass of macrobenthos was in Maduo County. The RDA analysis results indicated that the biodiversity of aquatic vascular plants and macrobenthos was shaped by multiple natural factors, including altitude, temperature, and precipitation, while showing a significant negative correlation with socio-economic factors such as population density and regional gross domestic product. This study provides a scientific basis for the conservation and ecological restoration of aquatic ecosystems in the Yellow River.