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SO 2 , a gas signaling molecule, can be produced endogenously in mitochondria. Its hydrolysate, HSO 3 − , plays a key role in food preservation, cardiovascular relaxation, and other fields, suggesting that it is important to achieve its detection. Here, based on the Michael addition mechanism, four hemicyanine dye fluorescent probes (ETN, ETB, STB, and EIB) were designed and synthesized for responding to HSO 3 − . We evaluated the reaction ability of different probes with HSO 3 − and tried to explain the reasons for the significantly different response effects between probes and HSO 3 − according to the structure−activity relationship. The influence of different substituents of probes on the properties of mitochondria-targeting was also discussed. Finally, we screened out ETN as the optimal HSO 3 − probe due to its high sensitivity, rapid reactivity, and good mitochondria-targeting, and it could sensitively respond to HSO 3 − in living cells. The LODs of ETN for HSO 3 − were calculated by both absorption and fluorescence methods, respectively, which were 2.727 and 0.823 μM. Our work provided valuable references for designing strategies and potential tools for response to SO 2 derivatives in biosystems.