The high levels of nitrate (NO3-) in the surface water have contributed to eutrophication and other eco-environmental damages worldwide. Although the excessive NO3- concentrations in rivers were often attributed to anthropogenic activities, some undisturbed or slightly disturbed rivers also had high NO3- levels. This study utilized multi-pronged approaches (i.e., river natural abundance isotopes, N-15-labeling techniques, and qPCR) to provide a comprehensive explanation of the reason for the high NO3- levels in a river draining forest-dominated terrene. The river natural abundance isotopes (delta N-15/delta O-18-NO3-) indicated that the soil source (i.e., soil organic nitrogen-SON and chemical fertilizer-CF) were the primary contributors to the NO3-, and the NO3- removal was probably prevalent in the basin scale. The N-15-labeling techniques quantitatively showed that denitrification and anammox were stronger than nitrification in the soils and sediments. Structural equation models suggested that nitrification in the soils was regulated by NH4+-N contents, which, in turn, were closely related to fertilization in spring. Denitrification and anammox were largely controlled by elevation and functional gene abundances (i.e., nirK and hzsB, respectively). The hydrological isotopes (i.e., delta D/delta O-18-H2O) indicated that the transport of NO3- from soil to the river was related to the intensity of runoff leaching to the soil, In contrast, the riverine NH4+ was largely from point sources; thus, increasing runoff led to a dilution effect. This study clearly showed that soil biogeochemistry and hydrological condition of a river basin jointly shaped the high NO3- levels in the almost undisturbed river.