Sensing of intracellular singlet oxygen (O) is required in order to optimize photodynamic therapy (PDT). An optical nanoprobe is reported here for the optical determination of intracellular O. The probe consists of a porous particle core doped with the commercial O probe 1,3-diphenylisobenzofuran (DPBF) and a layer of poly-L-lysine. The nanoparticle probes have a particle size of ~80 nm in diameter, exhibit good biocompatibility, improved photostability and high sensitivity for O in both absorbance (peak at 420 nm) and fluorescence (with excitation/emission peaks at 405/458 nm). Nanoprobes doped with 20% of DPBF are best suited even though they suffer from concentration quenching of fluorescence. In comparison with the commercial fluorescent O probe SOSG, 20%-doped DPBF-NPs (aged) shows higher sensitivity for O generated at an early stage. The best nanoprobes were used to real-time monitor the PDT-triggered generation of O inside live cells, and the generation rate is found to depend on the supply of intracellular oxygen. Graphical abstract A fluorescent nanoprobe featured with refined selectivity and improved sensitivity towards O was prepared from the absorption-based probe DBPF and used to real-time monitoring of the generation of intracellular O produced during PDT.