Singlet oxygen (O-1(2)) detection remains an active area of research in the field of biological and biomedical science. In this work, we reported a novel Off-On type FRET-based fluorescent O-1(2) nanoprobe comprising the energy-donating conjugated polymer, poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO), and the energy-accepting O-1(2) indicator, 3-diphenylisobenzofuran (DPBF). Prior to the reaction with O-1(2), the nanoprobe was in Off-state because fluorescence of PFO (lambda(em)=441 nm) was completely quenched by loaded DPBF; in the presence of O-1(2), it switched to On-state due to the depletion of DPBF. The as-prepared O-1(2) nanoprobe had a particle size of similar to 59 nm, good biocompatibility, high specificity and sensitivity with a limit of detection of 353 nM. Importantly, the fluorescent nanoprobes were highly sensitive to O-1(2) under two-photon excitation (lambda(ex)=800 nm). Using a multiphoton confocal microscope, generation of intracellular O-1(2) during photodynamic therapy was sensitively detected by recording fluorescence intensity of the nanoprobes.