Superoxide anion (O 2 ·− ) is the primary species of the reactive oxygen species (ROS), resulting from the metabolism of O 2 in the living organisms. Its dynamic changes can provide broad implications in physiological and pathological conditions. Therefore, the analytical methods that can determine O 2 ·− real-time and in vivo are receiving more and more attention. In light of these requirements, electrochemical approaches are the most promising candidate techniques in analysis of O 2 ·− due to their simplicity, direct measurements, and ease of miniaturation. This review summarizes the recent progress on electrochemical analysis of O 2 ·− taking into consideration toward the key issues: design of the electrolyte/electrode interface, direct electron transfer of enzymes, and applications in live cells and animals.