Increased production of reactive nitrogen (Nr) from atmospheric di-nitrogen (N₂) has greatly contributed to increased food production. However, enriching the biosphere with Nr has also caused a series of negative effects on global ecosystems, especially aquatic ecosystems. The main pathway converting Nr back into the atmospheric N₂ pool is the last step in the denitrification process. Despite several attempts, there is still a need for perturbation-free methods for measuring in situ N₂ fluxes from denitrification in aquatic ecosystems at the field scale. Such a method is needed to comprehensively quantify the N₂ fluxes from aquatic ecosystems. Here we observed linear relationships between the δ¹⁵N-N₂O signatures and the logarithmically transformed N₂O/(N₂+N₂O) emission ratios. Through independent measurements, we verified that the perturbation-free N₂ flux from denitrification in nitrate-rich aquatic ecosystems can be inferred from these linear relationships. Our method allowed the determination of field-scale in situ N₂ fluxes from nitrate-rich aquatic ecosystems both with and without overlaying water. The perturbation-free in situ N₂ fluxes observed by the new method were almost one order of magnitude higher than those by the sediment core method. The ability of aquatic ecosystems to remove Nr may previously have been severely underestimated.