Hypoxia is a common feature of many diseases such as solid tumors. The measurement and imaging of oxygen (O 2 ) are extremely important for disease diagnosis and therapy evaluation. In this work, the afterglow/fluorescence dual-emissive ratiometric O 2 probe based on a photochemical reaction-based afterglow system is reported. The afterglow is highly sensitive to O 2 because the O 2 content is directly related to the 1 O 2 yield and eventually affects the afterglow intensity. The O 2 -insensitive fluorescence of an emitter can serve as an internal reference. As the O 2 concentration changes from 0.08 to 18.5 mg L -1 , the ratio value shows a remarkable 53-fold increase. Compared with the intensity of a single peak, the ratiometric signal can eliminate the interference of the probe concentration to achieve higher accuracy. This afterglow/fluorescence dual-emissive ratiometric O 2 probe is successfully applied to hypoxia imaging in tumor-bearing mice, which may further promote the development of O 2 sensing in the biomedical field.