Photodynamic therapy (PDT), which uses photosensitizers to produce singlet oxygen to kill bacteria, was first discovered by scientists over 100 years ago and has been widely used in clinical treatment. Riboflavin is an endogenous and the most commonly used photosensitizer.A large number of studies have shown that riboflavin can produce singlet oxygen through photodynamic reaction(typeⅡ) after illumination.This method is currently widely used to kill pathogens such as bacteria and viruses in blood, and singlet oxygen is considered to be the key factor in the photodynamic effect.Singlet oxygen can induce cell apoptosis, necrosis and autophagy.At present, there are direct and indirect methods to detect singlet oxygen, but both with some limitations.This paper reviews production mechanism, action mechanism and detection of singlet oxygen produced by riboflavin during photodynamic therapy, which provides a basis for further application of photodynamic therapy in clinical practice and searching for a more suitable detection method for singlet oxygen.