The simple, sensitive, and specific detection of hydrogen sulfide (H2S) is of great importance because of its crucial role in food safety, environmental pollution, and various pathological and physiological processes. Here, we reported activatable fluorescence nanoprobe-based quantum dots (QDs) for sensitive and selective monitoring of H2S in red wine, environmental water samples, and lysosome of live cancer cells. The nanoprobe was prepared through a strong electrostatic interaction between thioglycolic-acid-stabilized CdTe QDs and p-amino thiophenol capped silver nanoparticles (AgNPs) that resulted in the formation of the assembled nanostructure, called QD/AgNP nanocomplexes. The initial fluorescence of QDs was effectively quenched by the AgNPs because of the inner filter effect. Upon interaction with H2S, the strong etching ability of H2S to AgNPs could trigger the disassembly of QD/AgNP nanocomplexes and generate Ag2S on the surface of QDs, achieving a shell-core Ag2S/CdTe QDs with remarkable fluorescence as a result of the termination of inner filter effect. The aqueous solution studies displayed that the assembled QD/AgNP nanoprobe was sensitive to detect H2S, with a detection limit of 15 nM. In addition, this assembled QD/AgNP nanoprobe showed a high specificity toward H2S over other anions and biologically relevant species. The subsequent fluorescence imaging studies demonstrated that the assembled QD/AgNP nanoprobe exhibited high ability to enter into cellular lysosome and generated an enhancement fluorescence, which was used for endogenous H2S detection in lysosome of living cancer cells. This proposed nanoprobe revealed a more simple, rapid, time-saving, low-cost, sensitive, and selective process for monitoring of H2S in further environmental pollution, food safety, and clinical diagnosis of H2S-related diseases.