To understand the sources and transformations of nitrate in the Songhua River basin, which is one of seven largest river basins in China, the concentration of dissolved nitrogenous species, nitrogen and oxygen isotopes of NO₃⁻, nitrogen isotopes of NH₄⁺, and stable isotopes of water were determined in this study. Low NO₃⁻ concentrations and a high dissolved organic nitrogen/total dissolved nitrogen ratio (DON/TDN) were observed in the Nen River and other rivers originating from the mountains, which are covered by forest. NO₃⁻ and DON were the major nitrogenous compounds in aquatic systems, accounting for the TDN being about 90% during high flow season and about 85% during low flow season, respectively. The nitrogen efflux for the entire basin was estimated to be approximately 1.17 × 10⁵ tons/yr, which represents an annual N output of 0.21 ton/(km² yr). The majority of the δ¹⁸O-NO₃⁻ values were between −4‰ and 4‰, reflecting nitrification. During the high flow season, the isotopic compositions of NO₃⁻ and the water chemistry suggest that NO₃⁻ in the Nen River was mainly derived from soil organic nitrogen (SON), whereas NO₃⁻ in the Songhua River originated from organic nitrogen, nitrogenous fertilizers and sewage waters. NO₃⁻ in the low flow season samples generally originates from SON and sewage waters. Moreover, the calculated loss of nitrate via the mass budget in rivers, together with isotopic values and water chemistry confirm that denitrification occurs during the high flow season, especially in the Songhua River. This study suggests that the mass calculation and isotopic proof provide a better understanding for riverine N budget and biogeochemical processes.