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