A lanthanide complex-based ratiometric luminescence probe, [4′-(2,4-dinitrobenzenesulfonyloxy)-2,2′:6′, 2′′-terpyridine-6,6′′-diyl] bis(methylenenitrilo) tetrakis(acetate)-Eu3+/Tb3+ (NSTTA-Eu3+/Tb 3+), has been designed and synthesized for the specific recognition and time-gated luminescence detection of biothiols in physiological pH aqueous media. The probe itself is almost nonluminescent due to the presence of photoinduced electron transfer (PET) from the terpyridine-Ln3+ moiety to the 2,4-dinitrobenzenesulfonyl (DNBS) moiety. In the presence of biothiols, the reaction of NSTTA-Eu3+/Tb3+ with biothiols results in the cleavage of DNBS to afford the deprotonated (4′-hydroxy-2,2′: 6′,2′′-terpyridine-6,6′′-diyl) bis(methylenenitrilo) tetrakis(acetate)-Eu3+/Tb3+ (HTTA-Eu3+/Tb3+), which terminates the PET process. After the reaction, the intensity of Eu3+ emission at 610 nm is unchanged, while that of Tb3+ emission at 540 nm is remarkably increased, which provides a ∼36-fold enhanced intensity ratio of Tb3+ emission to Eu3+ emission (I540/I610). This unique luminescence response allows NSTTA-Eu3+/Tb3+ to be used as a ratiometric probe for the time-gated luminescence detection of biothiols, using the intensity ratio of I540/I610 as a signal. Thus, based on the probe NSTTA-Eu3+/Tb3+, a ratiometric time-gated luminescence detection method for biothiols was established and successfully used for the quantitative detection of the total biothiols in several living cell samples.