Hydrological fluctuations drive community dynamics in floodplain lakes, yet their integration into water resource management remains limited. Here, we integrated environmental DNA (eDNA) metabarcoding with hydroacoustic surveys to investigate vertebrate community assembly in China’s largest freshwater lake (Poyang Lake) during the winter dry season. We detected 65 vertebrate species, with Cypriniformes dominating. Beta-diversity partitioning revealed that turnover dominated taxonomic and functional dissimilarity, while phylogenetic beta diversity was characterized by nestedness, which is consistent with environmental filtering. Functional richness declined with water depth, coinciding with hydroacoustic vertical size stratification, indicating niche partitioning along depth gradients. Null model analysis showed stochastic processes (ecological drift) dominated regional assembly (72.97%), whereas joint species distribution modeling attributed explained variation to environmental factors (28.9%, notably water depth) and species associations (29.7%) at local scales. This hierarchical framework, regional stochasticity shaping the species pool and local deterministic filtering structuring communities, reframes environmental flow conceptualization: effective management must preserve the full spectrum of hydrological variability and maintain bathymetrically diverse habitats that support functional niche differentiation. The integrated eDNA-hydroacoustic approach offers a non-invasive, high-resolution toolkit for biological assessment within regulatory water quality frameworks.