Abstract: Global aquatic deoxygenation is reshaping carbon cycling in inland waters, yet how oxygen loss regulates dissolved organic matter (DOM) and microbial linkages, as well as refractory DOM formation in seasonally stratified reservoirs, remains not fully examined. Here, we combined seasonal hydrochemical observations with optical spectroscopy, high resolution mass spectrometry, and 16S rRNA gene sequencing to examine DOM composition and bacterial community responses along stratification induced oxygen gradients in a subtropical river-reservoir continuum in southeast China. Strong summer stratification produced a pronounced oxycline and hypoxic hypolimnion, creating steep gradients in oxygen availability and microbial niches. Along this gradient, FDOM_P, UA+Peptides compounds decreased, whereas FDOM_H, AI_mod, CA+Poly, CRAM, and I_DEG increased, indicating microbial conversion of labile DOM into oxidized refractory components. The oxycline showed the highest depth normalized accumulation of FDOM_H, 10.3 times higher than in the epilimnion and 1.9 times higher than in the hypolimnion after excluding density current effects, identifying this layer as the main zone of in situ RDOM production. This enrichment was probably supported by the joint availability of energy rich bio labile substrates and oxygen supplied from the upper water column, which promoted microbial oxidation of substrates with high activation energy. Bacterial richness, diversity, evenness, and CRAM chemodiversity also peaked in the oxycline, suggesting that redox niche differentiation and metabolic complementarity contributed to the diversification of refractory DOM. Across the river reservoir continuum, dam driven changes in DOM quality and bacterial community structure reshaped DOM and bacteria interaction patterns, with consequences for carbon transformation, persistence, and downstream transport. Together, these findings identify reservoir oxyclines as active zones of microbial carbon processing and highlight dam-driven oxygen restructuring as an important control on DOM persistence and carbon transfer across river-reservoir continuum.