The disturbance of reactive nitrogen (N) on ecosystems and biogeochemical cycles is now one of the most severe environmental problems worldwide. Nitrate (NO3−) is usually a dominant reactive N species in river ecosystems. Excessive NO3− concentrations in rivers have led to eutrophication and consequent ecological and environmental damages. Quantifying catchment-scale NO3− yield and export dynamics is crucial for effective remediation of river NO3− pollution. Frequently, natural abundance isotopes of NO3− in a river (δ15N/δ18O-NO3−) are applied to identify sources and potential transformations of NO3− at a catchment scale, while microbial molecular techniques and 15N pairing experiments are employed to reveal the NO3− production and removal processes and their underlying mechanisms in microenvironments (e.g., sediments and soils). In this study, we developed a novel protocol that couples these complementary geochemical and molecular techniques to quantify catchment-scale NO3− yield and fluvial export dynamics. The protocol links microscopic processes with catchment-scale geochemical characteristics to explicitly describe the NO3− cycling processes and their underlying abiotic and biotic mechanisms within a catchment. We applied the protocol to the Dadu and Jiazela catchments on the Qinghai-Tibet Plateau, and demonstrated the effectiveness of the protocol in determining NO3− yield and export dynamics in the catchments.