Water depth variation can lead to the vertical structure change of microbial communities in reservoirs, and then affect the relationship between the microbial communities along the depth gradient, profoundly affecting the stability of the aquatic ecosystems. However, the interspecific dynamics of microbial communities across different water layers in deep-water low-nutrient drinking water reservoirs remain not well understood. Thus, we assessed microbial communities' dynamic changes in different water layers in this study. The physical and chemical parameters and different planktonic microbial of the surface, middle, and bottom layers were studied from July 2022 to August 2023 in the Danjiangkou Reservoir, China. Based on high-throughput sequencing technology, model analysis and network analysis, the diversity of microbial communities in different water layers, community construction process and co-occurrence network differences were studied. The results showed that the diversity of bacterial communities in the Danjiangkou reservoir was significantly higher than that of fungi and eukaryotic microorganisms in different water depths. The dominant taxa of the bacterial communities in different water depths were Actinobacteriota, Bacteroidota, Proteobacteria and Cyanobacteria. The dominant phyla were Ascomycota, unclassified_k__Fungi and Chytridiomycota. The relative abundance of vertical dominant species in eukaryotic communities was slightly different, including Cryptophyta, Chlorophyta, Dinophyta and Metazoa. Different microbial communities shared the main dominant species on the vertical stratification. The neutral model showed that random processes significantly affected the assembly process of microbial communities in different water layers, and the mobility of fungal communities was much lower than that of bacteria and eukaryotes. The co-occurrence network analysis showed that the number of nodes and edges of the bacterial community was the highest, indicating that the network scale of the bacterial community was the largest. In addition, the map density and average clustering coefficient of bacterial and eukaryotic communities in surface water were the highest, indicating that the surface microbial species had a high degree of connectivity, can better transfer materials and exchange information, and Sensitive to changes in the external environment. In contrast, in fungal communities, microbial interactions were the most complex at the bottom. The interactions between microbial communities in different water depths were mainly positive, and the negative correlation of microbial communities in the middle and bottom water was greater than that in the surface water, indicating that the competition between species increased with the increase of depth. Correlation analysis showed that the key species of microbial community were significantly correlated with TP, PO