In this study, we present an innovative "click-to-release" strategy for the design of highly specific H2Sn bioorthogonal probes that undergo a specific click reaction with H2Sn and release fluorophores by a following rearrangement. A library of cyclooctyne derivatives was established and successfully demonstrated the availability of the release strategy. Then, a model probe CM-CT was synthesized, which can achieve effective fluorophore release (>80%) in the presence of a H2Sn donor. To further validate the application of this class of probes, a new probe QN-RHO-CT based on Rhodamine 110 was developed. This probe showed good water solubility (>160 μM) and fast release kinetics and can achieve selective H2Sn detection in living cells. We used this probe to study the process of H2S-mediated protein S-persulfidation and demonstrated that excess H2S would directly react with protein persulfides to generate H2S2 and reduce the persulfides to thiols. Additionally, we elucidated the click-to-release mechanism in our design through a detailed mechanistic study, confirming the generation of the key intermediate α, β-unsaturated cyclooctanethione. This bioorthogonal click-to-release reaction provides a useful tool for investigating the function of H2Sn and paves the way for biological studies on H2Sn.