As　a　topoisomerase　I　inhibitor,　the　prodrug　irinotecan　has　been　widely　used　in　the　chemotherapy　of　metastatic　cancers.　However,　the　transformation　from　irinotecan　to　its　active　metabolite　SN-38　is　slow　and　non-selective　in　cancer　cells,　which　is　not　desired　for　clinical　oncotherapy.　Additionally,　the　fluorescence　turn-on　response　of　irinotecan　is　poor,　prohibiting　real-time　observations　on　SN-38　release.　Here,　we　developed　a　new　prodrug　(BDD-SN38)　that　is　well　suited　for　real-time　monitoring　SN-38　release　and　reducing　its　activation　in　normal　cells.　The　fluorescence　and　activity　of　BDD-SN38　was　significantly　quenched　by　dinitrobenzene　group.　The　in　vitro　and　in　cell　results　showed　that　BDD-SN38　could　be　selectively　and　rapidly　activated　by　hydrogen　sulfide　(H2S)　to　obtain　significantly　enhanced　fluorescence　and　activity.　Moreover,　BDD-SN38　achieved　remarkable　toxicity　towards　H2S-overexpressed　cancer　cells.　Overall,　the　H2S-responsive　prodrug　developed　here　was　proved　to　be　a　prom-ising　candidate　for　real-time　monitoring　SN-38　release　and　improving　its　therapeutic　effect.