Abstract: AimTo establish a method for screening active substance with scavenging effects on superoxide anion in vitro by designed superoxide dismutase biosensor．MethodsThe enzyme sensor was built by connecting the immobilized CuZnSOD with optical oxygen sensor through a special way． Superoxide anions were generated by auto-oxidation of pyrogallol． The auto-oxidation speed was examined before and after adding samples into the system， and the Vit C having the scavenging radical activities was served as a positive control．ResultsThe limit of biosensor detection was 7.0 U in activity， and lifetime of the immobilized enzyme in the reaction-cell was above 2 weeks． The scavenging effects on superoxide radicals of fifteen active substance were studied in vitro by the sensor， and some of them presented scavenging activities． ConclusionThe signal from biosensor is stable， easy to be determined， and the kinetic information on scavenging superoxide radicals could be obtained directly． The biosensor system can be used for screening drugs simply and rapidly．