Display omitted •Molecular composition of water-extractable DOM was analyzed in two deep-sea sediment cores.•Microbial community composition was linked to DOM composition in the sediment cores.•Microbes play a crucial role in the distribution of sedimentary recalcitrant DOM.•Sediments are a potential source of deep-sea recalcitrant DOM. Sedimentary organic matter provides carbon substrates and energy sources for microorganisms, which drive benthic biogeochemical processes and in turn modify the quantity and quality of dissolved organic matter (DOM). However, the molecular composition and distribution of DOM and its interactions with microbes in deep-sea sediments remain poorly understood. Here, molecular composition of DOM and its relationship with microbes were analyzed in samples collected from two sediment cores (∼40 cm below the sea floor), at depths of 1157 and 2253 m from the South China Sea. Results show that niche differentiation was observed on a fine scale in different sediment layers, with Proteobacteria and Nitrososphaeria dominating the shallow sediments (0–6 cm) and Chloroflexi and Bathyarchaeia prevailing in deeper sediments (6–40 cm), indicating correspondence of microbial community composition with both geographical isolation and the availability of organic matter. An intimate link between the DOM composition and microbial community further indicates that, microbial mineralization of fresh organic matter in the shallow layer potentially resulted in the accumulation of recalcitrant DOM (RDOM), while relatively low abundance of RDOM was linked to anaerobic microbial utilization in deeper sediment layers. In addition, higher RDOM abundance in the overlying water, as compared to that in the surface sediment, suggests that sediment might be a source of deep-sea RDOM. These results emphasize the close relation between the distribution of sediment DOM and different microbial community, laying a foundation for understanding the complex dynamics of RDOM in deep-sea sediment and water column.