Dissolved organic matter (DOM) constitutes the largest active carbon pool on earth, playing a crucial role in numerous biogeochemical processes. Understanding the molecular characteristics and chemical properties of DOM is essential for comprehending the global carbon cycle. However, there is a lack of systematic understanding regarding the influence of periodic flooding and drying, caused by reservoir operations, on the sources, characteristics and stability of soil DOM in the drawdown area, as well as the biotic and abiotic processes regulating DOM changes. This study employs Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and 16S rRNA sequencing to investigate the variations in molecular and compound composition of soil DOM at different elevations in the drawdown area of the Three Gorges Reservoir, and their associations with microbial communities. The results indicate that with the increasing duration of flooding, the proportion of easily degradable DOM gradually increases in the drawdown area soils, while the proportion of refractory DOM decreases. Periodic flooding and drying enhance the microbial authigenic components of DOM, reduce the plant-derived DOM components, and significantly decrease the stability, aromaticity, and unsaturation of soil DOM. Soil DOM engages in the biogeochemical processes of the drawdown area ecosystem through coupled changes with bacteria and archaea, and changes in soil DOM result in variations in microbial necromass carbon and lignin phenol content at different elevations. The findings are significant for deepening the understanding of the biogeochemical processes involving soil DOM in drawdown areas.