A new europium(III) complex, [4'-(10-methyl-9-anthryl)-2,2': 6', 2"-terpyridine-6,6"-diyl] bis(methylenenitrilo) tetrakis(acetate)-Eu3+, was designed and synthesized as a highly sensitive and selective time-gated luminescence probe for singlet oxygen (O-1(2)). The new probe is highly water soluble with a large stability constant of similar to 10(21) and a wide pH available range (pH 3-10), and can specifically react with O-1(2) to form its endoperoxide (EP-MTTA-Eu3+) with a high reaction rate constant at 10(10) M-1 s(-1), accompanied by the remarkable increases of luminescence quantum yield from 0.90% to 13.8% and lifetime from 0.80 to 1.29 ms, respectively. The wide applicability of the probe was demonstrated by detection of O-1(2) generated from a MoO42-/H2O2 system, a photosensitization system of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)-porphyrin tetra(p-toluenesulfonate) (TMPyP), and a horseradish peroxidase catalyzed aerobic oxidation system of indole-3-acetic acid (IAA). In addition, it was found that the new probe could be easily transferred into living HeLa cells by incubation with TMPyP. A time-gated luminescence imaging technique that can fully eliminate the short-lived background fluorescence from TMPyP and cell components has been successfully developed for monitoring the time-dependent generation of O-1(2) in living cells.