A lysosome-specific fluorescent probe, Lyso-AC, for biothiols was developed by incorporation of a 4-nitrophenol moiety into a coumarin dye. The Cys/Hcy-triggered substitution-rearrangement cascade, and GSH-induced substitution reaction lead to the corresponding blue emissive amino-coumarin and yellow emissive thiol-coumarin, thereby enabling Cys/Hcy and GSH detection from distinct emissions. Moreover, this probe displayed an excellent lysosome targeting property with a 0.92 Pearson's colocalization coefficient by using Neutral Red as a reference. Significantly, biological experiments indicated Lyso-AC has the potential to monitor lysosome Cys/Hcy and GSH simultaneously in living HeLa cells from distinct emissions.