As significant players in material cycling and energy flow, bacteria and eukaryotes play a vital role in the ecosystem. Nevertheless, the community dynamics of bacteria and eukaryotes in rivers and their responses to changes in ecological hydrology have not been studied thoroughly. Based on eDNA technology, this study investigated the bacterial and eukaryotic communities in the upper, middle and lower reaches of the Weihe River in different seasons. The seasonal variation and geographical distribution of bacterial and eukaryotic community structures showed significant heterogeneity. The selective theory well explained the response of microbial community assembly to seasonal changes. Deterministic processes dominate microbial community assembly in the middle and lower reaches. The composition and metabolic potential of key functional genes of nitrogen and phosphorus cycling (nosZ, pqqB, pqqD, and pqqE) exhibited strong seasonal patterns and were significantly correlated with the physical and chemical properties of water. There were significant differences in molecular ecological networks in different periods (p < 0.05), with a gradually increasing trend in the complexity of the network from winter to summer. The keystone species (Hub) of the microbial food web in each season included microorganisms (Malikia), algae (Stephanodiscus), and invertebrates (Polyarthra). Structural equation modeling (SEM) results indicated that invertebrate was an important driving factor affecting the changes in community structures. In micro-food webs, both “bottom-up” (resources) and “top-down” (predation) forces strictly controlled the relationship between taxa. Nitrogen and phosphorus concentrations affected microbial networks, and there was a significant correlation between bacterial and eukaryotic groups and eco-hydrological variables (p < 0.05). Furthermore, we identified the taxon’s change point using threshold indicator taxa analysis (TITAN), quantitatively revealing the response thresholds of taxa to eco-hydrological changes.