A complicated relationship between the active small molecules exists in cells. On the organelle level, active small molecules also play an important role in the maintenance of organelle functions and roles. To investigate the relationship of biomolecules in subcellular, it is necessary and critical to develop molecular tools that can track two kinds of associated biomolecules within organelles with multiple fluorescence signals. However, this is still an unmet challenge up to date. Herein, we present the first single-fluorescent probe (Lyso-HA-HS) that can detect oxidative (HOCl) and reductive (H2S) substances within an organelle (lysosomes) with multiresponse signals. The reactions of the new probe with H2S and HOCl simultaneously result in the blue and red channels emissions, respectively, providing different signal responses to the oxidative and reductive substances in the cellular lysosomes. Using a single fluorescent probe, we first achieved dual-channel imaging of the endogenous hypochlorous acid and hydrogen sulfide, respectively, in the lysosomes in the living cells. Moreover, the highly desirable attributes of the probe Lyso-HA-HS (such as high selectivity, good membrane-permeability, and lysosome enrichment ability) may enable it to be used in revealing the relationship of HOCl and H2S in lysosomes.