A great number of fluorescent probes have been developed for detecting singlet oxygen (1O2), which is considered to be one of the most effective reactive oxygen species (ROS), especially in clinical applications. The commercially available fluorescent probe Singlet Oxygen Sensor Green (SOSG) is widely used due to its reported high selectivity to 1O2. In this study, we carried out systemic experiments to determine the activation of SOSG in the presence of ionizing radiation. The results show that the SOSG probe exhibits a pronounced fluorescence increase as a function of radiation dose delivered by gamma-rays as well as X-rays, in conditions where the formation of singlet oxygen is not expected. Furthermore, scavenger tests indicate that hydroxyl radicals may be involved directly or indirectly in the activation process of SOSG although the exact mechanism remains unknown.