In this paper, the powder microelectrode technique was employed in studying the voltametric response of the O2/2− couple, which demonstrated a nearly reversible redox process at an acetylene black powder microelectrode in N,N-dimethylformamide (DMF). A well-developed steady state current plateau for the electrochemical reduction of oxygen was obtained in this system. The electron transfer number (n) and heterogeneous electron transfer rate constant ks were measured by steady-state voltametric response, and the results were 1.08, 3.4 × 10−3 cm s−1, respectively. Additionally, the scavenging activity of O2− with biological antioxidant (ascorbic acid) was evaluated by cyclic voltammetry; IC50 came to 5 × 10−4 mol/l.