Nitrogen (N) fixation is the conversion of inert nitrogen gas (N₂) to bioavailable N essential for all forms of life. N₂-fixing microorganisms (diazotrophs), which play a key role in global N cycling, remain largely obscure because a large majority are uncultured. Direct probing of active diazotrophs in the environment is still a major challenge. Herein, a novel culture-independent single-cell approach combining resonance Raman (RR) spectroscopy with ¹⁵N₂ stable isotope probing (SIP) was developed to discern N_2-fixing bacteria in a complex soil community. Strong RR signals of cytochrome c (Cyt c, frequently present in diverse N_2-fixing bacteria), along with a marked ¹⁵N_2-induced Cyt c band shift, generated a highly distinguishable biomarker for N₂ fixation. ¹⁵N_2-induced shift was consistent well with 15N abundance in cell determined by isotope ratio mass spectroscopy. By applying this biomarker and Raman imaging, N_2-fixing bacteria in both artificial and complex soil communities were discerned and imaged at the single–cell level. The linear band shift of Cyt c versus ¹⁵N₂ percentage allowed quantification of N₂ fixation extent of diverse soil bacteria. This single-cell approach will advance the exploration of hitherto uncultured diazotrophs in diverse ecosystems.