Hydrogen sulfide (H2S) is an important molecule in cellular physiology, serving various functions such as regulating blood pressure, protecting cells against oxidative stress, modulating cellular metabolism, and regulating cell survival and death. Another essential biomolecule is hypochlorous acid (HClO), which is produced by white blood cells to eliminate bacteria and viruses during infection. However, excessive production of ClO– can lead to tissue damage and contribute to many chronic diseases. Several fluorescent probes have been developed for the sensitive and selective detection of HS– and ClO–, but most of them are designed to target only one of these analytes. Here we report a sequential detection mode fluorescence probes P1-P3 that allow for the sensing of HS– and ClO–. The mixture of P and HS– constructs a specific sensing system for ClO–, leading to significant fluorescent quenching. Mechanism studies demonstrate that HS– substitutes the Cl atom in P, which leads to fluorescence enhancement. Furthermore, the addition of ClO– facilitates a ring-forming reaction, resulting in the formation of a thiofuran ring within the product (T) that quenches the fluorescence. Interestingly, P has a highly ordered steric packing and could self-assemble into a rice-spike-like structure. Upon the addition of HS–, the assemblies decompose into free molecules. After interaction with ClO–, these molecules further transform to T with strong assembled capacity, featuring a larger number of nanosheets. This study provides a novel mechanism for sensing HS– and ClO–, cell and living animal imaging further indicating the good application prospects of these probes in biosensing and bioimaging.