Singlet oxygen (O-1(2)) plays a key role in the photodynamic therapy (PDT) technique of neoplastic diseases. In this work, by using a 9,10-dimethyl-2-anthryl-containing beta-diketone, 1,1,1,2,2-pentafluoro-5-(9',10'-dimethyl-2'-anthryl)-3,5-pentanedione (Hpfdap), as a O-1(2)-recognition ligand, a novel beta-diketonate- europium(III) complex that can act as a luminescence probe for O-1(2), [Eu(pfdap)(3)(tpy)] (tpy = 2,2',2 ''-terpridine), has been designed and synthesized for the time-gated luminescence detection of O-1(2) in living cells. The complex is weakly luminescent due to the quenching effect of 9,10-dimethyl-2-anthryl groups. After reaction with O-1(2), accompanied by the formation of endoperoxides of 9,10-dimethyl-2-atithryl groups, the luminescence quenching disappears, so that the long-lived luminescence of the europium(III) complex is switched on The complex showed highly selective luminescence response to O-1(2) with a remarkable luminescence enhancement. Combined with the time-gated luminescence imaging technique, the complex was successfully used as a luminescent probe for the monitoring of the time-dependent generation of O-1(2) in 5-aminolevulinic acid (a PDT drug) loaded HepG2 cells during the photodynamic process. In addition, by coloading the complex and a mitochondrial indicator, Mito-Tracker Green, into HepG2 cells, the specific localization of [Eu(pfdap)(3)(tpy)] molecules in mitochondria of HepG2 cells was demonstrated by confocal fluorescence imaging measurements.