Estuaries sit at the land‐ocean boundary and act as natural reactors for nitrogen (N) transformations among numerous forms. Superimposing onto physical mixing, biological processes lead to nonconservative N behavior in estuaries. Under the coinfluences of biological alteration and multiple end‐members, however, some N species can exhibit apparent conservation. To explore N dynamics and potential mechanisms that modulate the mixing behaviors, we measured concentrations and natural isotopic compositions of multiple N species (nitrate: NO₃⁻, nitrite: NO₂⁻, ammonium: NH₄⁺, and particulate nitrogen) in a typical eutrophic estuary in southern China. Additionally, N uptake and oxidation rates were measured by using isotope labelling techniques to evaluate processes potentially offsetting the conservative mixing of specific N pools. We found that NO₃⁻ followed conservative two end‐member mixing with dual isotopes varying in a narrow range (<1–2‰) due to low microbial preferences. Moreover, δ¹⁵N‐NO₂⁻ revealed a nonconservative pattern with an involvement of multiple end‐members. The dominant N transformation processes shifted downstream. In the upper estuary, ammonia oxidation (~20 μmol L⁻¹ day⁻¹) dominated NH₄⁺ removal and was accompanied by NO₂⁻ accumulation. In the middle‐lower estuary, NH₄⁺ uptake became dominant, with phytoplankton showing strong preference for it over NO₃⁻ and NO₂⁻. Based on measured NH₄⁺ uptake rates (9.1–12.5 μmol L⁻¹ day⁻¹ in the light and 0.9 μmol L⁻¹ day⁻¹ in the dark), short water residence time (<1 day) was required to maintain the conservative mixing. Coupling N isotopes in multiple nitrogen species with measurements of N uptake and nitrification, we successfully uncovered N dynamics and distinguished biological processes from physical mixing.