Hydrogen selenide (H₂Se), a central metabolite of Se supplements, displays critical biological functions in many physiological and pathological processes. To better understand its comprehensive function, especially those exerted in subcellular organelles, the development of specific assays is urgently needed. However, the methodology to detect H₂Se is poorly developed. Here, we present a concise design strategy to obtain an activatable fluorescent probe (Se-1) for H₂Se by utilizing an intramolecular photoinduced electron transfer (PET) process to switch the fluorescence. The probe is able to selectively react with H₂Se without interference from intracellular reactive species, and has been successfully used to image the H₂Se content in lysosomes. Additionally, with the aid of Se-1, we demonstrated that lysosomal H₂Se can be generated and can gradually accumulate in HepG2 cells under hypoxic conditions. These applications make Se-1 a potential new candidate for deciphering the biological effects of H₂Se on lysosomes in biology and pathology.