The drivers of community dynamics during dry seasons remain poorly understood in many aquatic ecosystems, especially in highly dynamic floodplain lakes. We integrated hydroacoustic profiling and environmental DNA (eDNA) metabarcoding to investigate winter fish community assembly mechanisms in Poyang Lake, China’s largest freshwater lake. Vertical stratification partitioned species by body size, with larger taxa (>70 cm total length, TL) dominating benthic zones and smaller individuals (<30 cm TL) aggregating in surface waters. Despite this vertical niche differentiation, horizontal homogenization of species composition (NMDS: ANOSIM R2 = 0.065, p = 0.2) reflected ecological drift due to resource competition (βNTI |RC| < 0.95). A strong correlation between water depth variation (CV) and fish density fluctuation (R² = 0.62; p < 0.01) highlighted habitat fragmentation effects. Furthermore, low phylogenetic turnover revealed phylogenetic niche conservatism within the regional species pool. Winter habitat fragmentation confined communities to profundal refugia, intensifying stochastic assembly (undominated processes: 72.97%; homogenizing dispersal: 26.15%) via niche contraction, which drove elevated taxonomic β-diversity (βSør= 0.634; βJacc= 0.776). These findings underscore the necessity of maintaining bathymetric heterogeneity and hydrological connectivity to mitigate stochastic dominance in Poyang Lake’s winter fishery management.