Understanding the sources and biogeochemical processing of dissolved organic matter (DOM) and nutrients within large rivers is vital to carbon cycling and water management. However, there remains great uncertainty regarding aquatic DOM composition and source proportioning resulting from spatial-temporal heterogeneity. In this study, multiple techniques were applied to track riverine DOM and nutrients over different flow regimes along the middle reach of the Yangtze River. Combined with nitrate dual isotopes, three different organic sources have been identified including terrestrial humic substances, “non-natural” terrestrial humic substances associated with the input of urban sewage, and tyrosine-like substances related to microbiological activities. The higher aromatic degree of DOM, higher \({{\delta }}^{18}{\text{O}}_{{\text{N}\text{O}}_{3}}\), and lighter \({{\delta }}^{15}{\text{N}}_{{\text{N}\text{O}}_{3}}\) signals in the rainy season suggested that soil erosion by intensive monsoon rainfall was the main factor for the release of carbon and nitrogen. During the dry season, nitrate from soil solution in the riparian zone with denitrification and inputs of urban sewage might be responsible for the enrichment of \({{\delta }}^{15}{\text{N}}_{{\text{N}\text{O}}_{3}}\) and higher tyrosine-like substances. At the spatial scale, the same terrestrial DOM was observed between the distal surface water in the Three Gorge Reservoir and the outlet, which was mainly caused by the density currents inducing water intrusion in the reservoir. An ever increasing proportion of tyrosine-like substances longitudinally indicated the cumulative intensity of human activities was becoming stronger along the mainstream. The study advances our evaluation of different carbon and nitrogen in sources in the Yangtze River and provides insights into the management of large monsoonal rivers.
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