Singlet oxygen (O) plays important roles in many biological processes. However, it is very difficult to detect O in the intracellular environment because of its relatively low concentration and short lifetime. Here, we developed a ratiometric probe based on semiconducting polymer dots (Pdots) that can sensitively detect O in live cells. An organic dye, singlet oxygen sensor green (SOSG), was doped in polyfluorene Pdots, and excitation energy was efficiently transferred from the polymer to the SOSG dye. Accordingly, the Pdots showed constant blue fluorescence as a reference, and increased green fluorescence upon singlet oxygen generation. The ratiometric response of Pdots was examined in the intracellular environment by in situ O generation with a photosensitizer and light irradiation. Both spectroscopic measurements and confocal imaging were performed to monitor intracellular O generation during photodynamic therapy using the Pdot probe. Our results indicate that the SOSG-doped Pdots are promising for intracellular O detection.