Nitrogen is an essential nutrient element sustaining all life forms. Environmental microorganisms play an important role in biogeochemical cycle of nitrogen. However, because a large majority of environmental microorganisms are uncultured, our knowledge on them is very limited. Here, by combining surface-enhanced Raman spectroscopy (SERS) with 15N stable isotope probing, obvious 15N-induced band shifts in SERS spectra of bacteria were identified. Bands shifted linearly with 15N percentage in media, providing a way to study extent of nitrogen assimilation or activity of bacteria. The selective enhancement of SERS on N-related biomolecules accounted for the presence of indicator bands. Finally, SERS band shift induced by 15N, 13C and deuterium were compared and the results indicated that 15N induced a larger shift than deuterium. The reason is related to the position of N and H in the ring structure of adenine.