A direct and rapid SDS-PAGE staining method for in situ identification of activity and molecular weight of superoxide dismutase following denaturing treatment has been developed. This technique was based on the removal of SDS after SDS-PAGE and two-step staining procedures of the SDS-polyacrylamide gel to present the achromatic activity-zones of the enzymes. We demonstrated that the detection sensitivity of SDS-PAGE staining method was the same as the traditional xanthine oxidase-NBT solution assay. Through the SDS-PAGE staining method, three classes of superoxide dismutases with distinct molecular sizes were identified in situ. Moreover, activity of copper and zinc containing superoxide dismutase in crude extracts of Escherichia coli and Actinobacillus pleuropneumoniae was significantly enhanced using the two-step staining procedure.