Microbe-mediated biogeochemical cycle of nitrogen is a critical process in the environment. In this study, surface-enhanced Raman spectroscopy combined with ¹⁵N stable isotope probing (SERS–¹⁵N SIP) was developed as a new, nondestructive, and robust approach to probe nitrogen assimilation by bacteria at both bulk and single-cell level, and from pure culture to environmental microbial community. Multiple distinguishable SERS band shifts were observed and displayed a linear relationship with ¹⁵N content, because of the substitution of “light” nitrogen by “heavier” ¹⁵N stable isotope. These shifts, especially in 730 cm^–1 band, were highly distinguishable and universal in different bacteria, providing a robust indicator for nitrogen assimilation in bacteria. SERS–¹⁵N SIP was also demonstrated in important N₂-fixing bacteria via ¹⁵N₂ incubations. The same prominent shifts as that induced by ¹⁵NH₄Cl were observed, indicating the applicability of SERS–¹⁵N SIP to different nitrogen sources. SERS–¹⁵N SIP was further applied to environmental microbial community via ¹⁵NH₄Cl, ¹⁵NO^3–, and ¹⁵N₂ incubation. Bacteria- and nitrogen source-dependent activity in nitrogen assimilation were revealed in environmental microbial community, pointing to the bacterial diversity and necessity of single-cell level investigation. Finally, by mixing optimized ratio of bacteria with Ag NPs, explicit single-cell SERS–¹⁵N SIP was obtained. The nondestructive SERS–¹⁵N SIP approach will be useful not only to identify active nitrogen-assimilating cells, but also enable Raman activated cell sorting and downstream genomic analysis, which will bring in deep insights into nitrogen metabolism of environmental microorganisms.