Reactive oxygen species (ROS) play an essential role in regulating various physiological functions of living organisms. Superoxide anion (O2-.), one kind of ROS, is the single-electron reduction product of oxygen molecules, which mainly exists in plants and animals, and is closely related to many inflammatory diseases. In the field of biomedicine, with the deepening understanding of superoxide anion, more and more detection methods have been developed. This review mainly introduces the detection techniques for superoxide anion in recent years.