The direct electron transfer of superoxide dismutase (SOD) was greatly facilitated by sodium alginate (SA) sol–gel film with the formal potential of 0.14V, which was just located between O2−/O2 and O2−/H2O2. The preparation of the SOD/SA modified electrode was simple without any mediators or promoters. Based on bimolecular recognition for specific reactivity of SOD/SA toward O2−, the SOD modified electrode was utilized to measure O2− with good analytical performance, such as low applied potential (0V), high selectivity (no obvious interference), wide linear range (0.44–229.88μM) and low detection limit (0.23μM) in pH 7.0 phosphate buffer solution. Furthermore, it could be successfully exploited for the determination of O2− released from living cells directly adhered on the modified electrode surface. Thus, the proposed O2− biosensor, combining with the properties of SA sol–gel film, provided a novel approach for protein immobilization, direct electron transfer study of the immobilized protein and real-time determination of O2− released from living cells.