The nitrate (NO3–) dynamics on the Tibetan Plateau, one of the most climatic sensitive regions on Earth, has not yet been well understood. This study synthesized natural-abundance isotopes, 15N pairing techniques, and microbial functional genes to systematically elucidate the NO3− cycling dynamics as well as their biotic and abiotic driving mechanisms in a pristine forested catchment on the Tibetan Plateau. The river's natural-abundance isotopes showed that mineralization-nitrification of soil organic nitrogen was the major source of NO3− in the river, while significant NO3− removal occurred in the catchment. The 15N pairing experiments support the river's isotopes and quantitatively showed that nitrification was prevalent in soil, but more than half of the nitrification-derived NO3− was removed. In turn, the river isotopes verified the representativeness of the in-soil processes in the catchment. Structural equation models suggested nitrification and denitrification (the major NO3− removal pathway) were largely regulated by microbial functional gene abundances, which in turn were regulated by varying abiotic factors. In addition, the fluvial NO3– export rates in the sub-catchments were significantly correlated with the river runoff, highlighting the role of hydrological conditions and associated soil leaching in regulating fluvial NO3– fluxes. This study shows that combining multi-disciplinary techniques can achieve a more comprehensive understanding of NO3− dynamics at the catchment scale.