A study on the antioxidant activity of Portulaca oleracea L. in vitro was carried out by HPLC-ESR Spin-trapping System. HPLC-ESR analysis is performed on monitoring ESR signal intensity of radicals adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO/O2). The ESR signal which is recorded from the Portulaca oleracea L. extracts in H2O buffer by HPLC-ESR system showed that the high antioxidant activity of the extracts reduce the concentration of DMPO/O2 signal to 80% and the absorbance of reactive oxygen species from 0,08 to 0,06. The ground powder and the extracts of Portulaca oleracea L. were in vivo performed on GMR-GAL4; UAS-hDuox2 flies containing hDuox2 protein which induces high oxidative stress and expresses rough-eye phenotype. Antioxidant activities of Portulaca oleracea L. were evaluated by comparing rough-eye area before and after the experiment. At the concentration of 20%, the ground powder and the extracts induce antioxidant activities to 81.72% and 87.33% respectively. The result shows that both the ground powder and the extracts have antioxidant activities which reduce symptoms of rough-eye phenotypes. Especially, the extract is more effectively antioxidant than the ground powder.