Biothiols including cysteine (Cys), reduced glutathione (GSH), and hydrogen sulfide (H 2 S) play critical roles in living organisms. New reaction-based fluorescent turn-on probes based on fast thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) ether were explored for sensing of biothiols in aqueous buffer. The probes showed fast response toward biothiols with more than 100-fold off–on signal enhancement. Moreover, the probes were highly selective toward thiols over other amino acids and could be used for bioimaging in mammalian cells.