A terbium(III) chelate fluorescence probe for detection of singlet oxygen (1O2) in aqueous media, N,N,N1,N1-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-4-(9″-anthryl)pyridine] tetrakis (acetate)–Tb3+ (PATA-Tb3+), was designed and synthesized. The new chelate is highly water soluble, is almost nonfluorescent, and can specifically react with 1O2 to yield a strongly fluorescent chelate, the endoperoxide of PATA-Tb3+, accompanied by a remarkable increase in the fluorescence quantum yield from 0.46 to 10.5%. The long fluorescence lifetime of the endoperoxide (2.76 ms) allows the probe to be used favorably for time-resolved fluorescence detection of 1O2. The studies of fluorescence property and reaction specificity indicate that the new probe is highly sensitive and selective for 1O2. The probe was used for quantitative detection of 1O2 generated from a MoO42−–H2O2 system to give a detection limit of 10.8 nM. In addition, the good applicability of the probe was demonstrated by the real-time monitoring of the kinetic process of 1O2 generation in a horseradish peroxidase-catalyzed oxidation system of indole-3-acetic acid in a weakly acidic buffer.