Online detection and accurate quantification of superoxide anions released from skeletal muscle tissue is important in both physiological and pathological contexts. Above certain physiologically redundant levels, superoxides may exert toxic effects. Here we present design, fabrication, and successful testing of a highly sensitive electrochemical superoxide biosensor based on nanoporous gold (NPG) immobilized with cytochrome-c (cyt-c). A significant 14-fold enhancement in the biosensor sensitivity was achieved using NPG instead of nonporous gold, enabling the device to quantify minuscule levels of superoxides. Such improvement was attributed to the very large surface-to-volume ratio of the NPG network. The average values of superoxide sensitivity and analytical limit of detection (LOD) were 1.90 ± 0.492 nA nM^–1 cm^–2 and 3.7 nM, respectively. The sensor was employed to measure the rates of superoxide release from C2C12 myoblasts and differentiated myotubes upon stimulation with an endogenous superoxide-producing drug. To account for the issue of sensor-to-sensor sensitivity variations, each sensor was individually calibrated prior to measurements of biologically released superoxides. For the drug concentrations studied, C2C12 superoxide generation rates varied from 0.03 to 0.2 pM min^–1 cell^–1, within the range of superoxide release rates from normally contracting to fatiguing skeletal muscle tissue. Electrochemically obtained results were validated using a fluorescent superoxide probe. Compared to other destructive methods, the NPG-based electrochemical biosensor provides unique advantages in tissue engineering because of its higher sensitivity and the ability to measure the levels of biologically released superoxides in real-time.