Dissolved organic matter (DOM) is an essential component of the global carbon cycle, and estuaries link the rivers and oceans, thus playing important roles in land-ocean DOM transformation and transport. Hypoxia is a common phenomenon in estuaries and coastal oceans; however, its effects on DOM transport and fate remain unclear. To elucidate the effects of hypoxia on DOM characteristics, the molecular composition of DOM from bottom water and sediment porewater in hypoxic and non-hypoxic sites of the Yangtze River Estuary (YRE) was characterized utilizing ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry. The results significant differences in DOM molecular composition between hypoxic and non-hypoxic areas, for either bottom water or sediment porewater. There were lower H/C and molecular lability boundary index calculated by intensity (RA_MLBL), higher O/C, double bond equivalent (DBE), and modified aromaticity index (AI_mod) in hypoxic sites than in non-hypoxic oceanic sites, for either bottom water or sediment porewater, suggesting DOM in hypoxic areas was more recalcitrant than DOM in non-hypoxic areas. In addition, higher tannins, polyphenols, and black carbon in hypoxic areas suggested that hypoxic conditions could facilitate the preservation of terrestrial organic matter. Further, site-unique formulas were obtained by comparing common formulas in each site group (river, ocean-non-hypoxia, and hypoxia). The results showed a much higher number of hypoxia-unique formulas than ocean-non-hypoxia-unique formulas, indicating that hypoxia could diversify the DOM complexity. Besides, both biologically labile (unsaturated aliphatic compounds (UA) and peptides) and recalcitrant formulas (highly unsaturated compounds and carboxyl-rich alicyclic molecules) were found within hypoxia-unique formulas for sediment porewater. In contrast, most of the non-hypoxia formulas in sediment porewater were labile formulas, suggesting that hypoxia would facilitate the preservation of labile formulas and the production of recalcitrant formulas. Moreover, a much higher percentage of sulfur-containing formulas were found in hypoxia-unique formulas, especially in UA, indicating that sulfurization could be an important mechanism for the preservation of labile formulas. Therefore, our study could give insight into the effect of hypoxia on the molecular characteristics and preservation of DOM in estuaries and coastal oceans.