Marine nitrogen fixation contributes to the budget of biologically available N, thus fuels phytoplankton productivity and carbon cycle through biological pump. Modern N‐fixation rates are proved to be constrained by oceanographic condition and nutrient supply to the surface waters. However, the paleoceanographic reconstruction of N‐fixation and its regulation mechanism remain highly uncertain in many regions. Here we present records of N‐fixation changes in the South China Sea (SCS) over the past 250,000 years reconstructed by compound‐specific nitrogen isotopes of individual amino acids. The δ15N of source amino acids (δ15NSrc), reflecting the δ15N of the substrate nitrate originating from the subsurface water, is distinctly lower during interglacial periods, indicating intensified N‐fixation during interglacials. The δ15NSrc of the SCS co‐varies with the thermal gradient between surface and subsurface waters, implying a tight link between the upper water structure and N‐fixation. It could be hypothesized that stronger mixing during interglacials enhances the supply of excess phosphorous from the subsurface waters, thus encourages the growth of diazotrophs. Furthermore, records of bulk sediment δ15N with relatively high time resolution show dominant precession cycle, probably related to the nutrient supply from subsurface water driven by summer monsoon and associated upper water structure changes. Similar mechanism controlling N‐fixation is also effective in regions with enough iron supply and low concentrations of nitrogen and phosphorous, like the North Atlantic, supporting that upper water structure can dominate N fixation rates by regulating nutrient stoichiometry supplied to the surface waters.