The quantitative contribution of non-cyanobacterial diazotrophs (NCDs) to marine dinitrogen (N₂) fixation remains poorly constrained, limiting our understanding of the marine nitrogen cycle. To address this gap, we developed a pre-dawn dark-incubation approach that takes advantage of diel differences in diazotroph activity to minimize residual cyanobacterial N₂ fixation and provide an operational estimate of NCD N₂ fixation. Applying this method in the South China Sea, we found that NCDs represented a substantial component of euphotic-zone N₂ fixation, with their relative contribution generally increasing with depth. By integrating these field measurements with global environmental datasets using a boosted tree model, we further show that NCD contributions to surface-ocean N₂ fixation are spatially heterogeneous, with elevated contributions in selected subtropical and high-latitude regions. Model results and experimental evidence together suggest that dissolved organic carbon availability and iron limitation may help define the ecological niche of NCDs. These findings highlight NCDs as an important but previously underrepresented component of marine N₂ fixation and emphasize the need to explicitly consider their distinct biogeochemical controls in future marine ecosystem and climate models.