Molecular tools capable of providing information on a target analyte in an organelle of interest are especially appreciated. Traditionally, organelle-targetable probes are designed by incorporating an organelle-specific guiding unit to target the probe molecules into the organelle. The imperfect targeting function of the guiding unit and nonspecific distribution of the analyte in cytosol and each organelle would lead to low spatiotemporal resolution and limited sensitivity. To solve this problem, we report herein a new approach for detection of a target analyte in a specific organelle by engineering a target and location dual-controlled molecular switch. For this proof-of-concept study, fluorescent detection of H2S in lysosomes was performed with a simultaneous H2S and proton-activatable probe based on the acidic environment of lysosomes. The new synthesized fluorescent sensor, "SulpHensor", which contains a spirolactam moiety to bind hydrogen protons and an azide group to react with H2S, displays highly sensitive and selective fluorescence response to H2S under lysosomal pH environment but is out of operation in neutral cytosol and other organelles. Fluorescence imaging shows that SulpHensor is membrane-permeable and suitable for visualization of both the exogenous and endogenous H2S in lysosomes of living cells. The good performance of our proposed approach for H2S sensing demonstrates that this strategy might open up new opportunities for the development of efficient subcellular molecular tools for bioanalytical and biomedical applications.