Dissolved organic matter (DOM) exchange in the sediment-water interface of estuaries is essential for the global elemental cycle. To clarify the interface DOM processing, this study applies optical techniques and ultrahigh-resolution mass spectrometry to assess DOM composition of surface sediment porewater and bottom (overlying) water across the Yangtze River Estuary (YRE). Results suggested that DOM exchange in the sediment-water interface mainly followed from sediment porewater to bottom water driven by a significant dissolved organic carbon concentration gradient and hydrodynamic force. We also characterized two porewater DOM sources, including microbial production and byproducts of processed sediments. High microbial activities resulted in the enrichment of protein-like fluorescent components and N-bearing compounds in porewater, potentially decreasing the oxygen concentration of bottom water due to the high lability. And the deamination of N-bearing compounds in the sediment-water interface could likely serve as a N-bearing nutrient source to bottom water. Moreover, due to sediment-specific features in different areas driven by hydrologic sorting and local phytoplankton supply, porewater DOM of muddy areas accumulated more aromatic substances from the degradation of terrestrial organic matter. The release and oxic transformation of oxygen-deficient aromatic compounds could contribute to the refractory carbon pool of estuarine water (carboxyl-rich alicyclic molecules, CRAM), modulating the quality of organic carbon mobilized from the land to the coastal ocean. Considering strong hydrodynamic force in numerous estuaries worldwide, DOM exchange and processing at the sediment-water interface has a meaningful influence on the biogeochemistry of estuarine water columns, which warrants further studies.