BackgroundBisulfite (HSO3−) serves as a bleaching agent, antioxidant, antimicrobial, and regulator of enzymatic reactions in biosystem. However, abnormal levels of bisulfite can be detrimental to health. Hypochlorous acid (HOCl), which acts as bioactive small molecules, is crucial for maintaining normal biological functions in living organisms. Disruption of its equilibrium can lead to oxidative stress and various diseases. Therefore, it's essential to monitor the fluctuations of HOCl and HSO3− at cellular and in vivo levels to study their physiological and pathological functions. ResultsThis study constructed a novel NIR bifunctional colorimetric fluorescent probe using thienocoumarin-indanedione structures to identify hypochlorite (ClO−) and bisulfite (HSO3−). By using CSO-IO to recognize HSO3− and HOCl, two distinct products were generated, displaying green and blue fluorescence, respectively. This property effectively allows for the simultaneous dual-functional detection of HSO3− (LOD: 113 nM) and HOCl (LOD: 43 nM). SignificanceIn this work, the biocompatible molecule CSO-IO has been effectively designed to detect HOCl/HSO3− in living cells and zebrafish. As a result, the dual-functional fluorescent probe has the potential to be utilized as a molecular tool to detect HSO3− derived compounds and HOCl simultaneously within the complex biological system.