The transformation of litter-derived dissolved organic matter (DOM) in soils is important for carbon cycling in terrestrial ecosystems. However, the linkage between root litter- and rhizosphere soil-derived DOM remains unclear. In this study, using ultrahigh-resolution mass spectrometry and metagenomics, we evaluated the DOM in paired roots and rhizosphere soils for herbaceous plants in a semiarid grassland and their biogeochemical processes. Analyses revealed a decoupling between root- and rhizosphere soil-derived DOM despite being directly attached, with a considerable loss of root-derived aliphatics and proteins, and production of highly unsaturated, aromatic, and carboxyl-rich compounds. From roots to rhizosphere soils, DOM shifted toward a more uniform molecular composition, which was likely the result of a more "specialized" utilization of root-derived DOM and a more "generalized" utilization of rhizosphere soil-derived DOM by the rhizosphere microbial community. Overall, DOM transformation at the root-soil interface occurred along two principal dimensions: 1) the dimension of "root-to-soil variation" with "lability" and "aromaticity" as two end members, and 2) the dimension of "interspecies variation" dominated by bulk and optical DOM components. These findings suggest that root- and rhizosphere soil-derived DOM constitute two distinct carbon sources for rhizosphere microbial communities and provide a framework for future investigations into DOM dynamics and ecosystem functioning at the plant-soil interface.