Superoxide anion (SOA) as a member of reactive oxygen species (ROS) group is involved in various physiological and pathological states. For instance, generation of SOA is known to increase with skeletal muscle contractile activity and fatigue. It is therefore important to selectively detect and accurately quantify the release of SOA within both physiological and pathological levels. We report fabrication and characterization of a cytochrome-c functionalized SOA biosensor built on commercially available miniaturized screen-printed electrodes made of gold microspheres. The device was first tested and calibrated in a xanthine/xanthine oxidase (XOD) system and then employed to detect SOA release from C2C12 myoblasts and myotubes upon stimulation with PMA.