Hydrogen sulfide (H2S), as the third endogenous gasotransmitter, is closely associated with various physiological and pathological processes, whereas many aspects of its functions remain unclear. Effective tools for the accurate detection of H2S in living organisms are urgently needed. We herein reported an internal standard assisted surface-enhanced Raman scattering (SERS) nanoprobe for ratiometric detection of H2S in vitro and in living cells based on the reduction of nitros with H2S. This nanoprobe consists of an internal standard (4-mercaptobenzonitrile, MPBN) embedded core-molecule-shell Au nanoflower (Au@MPBN@Au) as the high plasmonic active SERS substrate and the 4-nitrothiophenol (4-NTP) molecule immobilized on the surface as the H2S recognition unit. With the addition of H2S, the nitros peak (1329 cm-1) decreased. Meanwhile, three obvious new peaks appeared at 1139, 1387, and 1433 cm-1, which were related to the vibration of the dimerized product 4,4'-dimercaptoazobisbenzene (DMAB) of 4-aminothiophenol (4-ATP). However, the peak intensity at 2223 cm-1 derived from MPBN was not influenced by the outer environment. Thus, the H2S level was able to be determined based on the ratio of two peak intensities (I1139/I2223) with a detection limit as low as 0.24 μM. Notably, we have proved that SERS nanoprobe Au@MPBN@Au@4-NTP could ratiometrically image both the endogenous and exogenous H2S in living cells. We anticipate that Au@MPBN@Au@4-NTP could be applied for the study of H2S-related physiological function in the future.