Recently, we discovered the H₂S-mediated reductive cleavage of the CC bond, and applied this reaction to design probes for the detection of H₂S. To extensively elucidate the structure–reactivity relationship, our current work further investigated the effect of substitutes that connected to the CC bond on the H₂S-mediated reductive cleavage, wherein different kinds of electron-withdrawing groups (such as pyridine, bipyridine, terpyridine) and electron donating groups (such as carbazole, N,N-dimethylaniline and phenothiazine) were conjugated to the CC bond of interest. Experimental results and DFT calculations showed that the strength of the electron-donating and withdrawing substitutes could significantly affect the reductive cleavage of the CC bond. Unexpectedly, the reductive cleavage was not influenced by the change of the C(2) and C(3) positions in phenothiazine. On this basis, two probes (NPTZ-P1 and NPTZ-P2) with a C(3)-substituted phenothiazine were thus developed and successfully applied for sensing exogenous H₂S in living HeLa cells, which implied their potential in bioimaging. This study provides further understanding on the structure–reactivity relationship of the H₂S-mediated reductive cleavage of the CC bond, and is also valuable for exploring new reactions of the CC bond in organic synthesis.