Several studies reported the antioxidant activity of bifidobacteria using assays in vitro. In present study, the in vitro and in vivo antioxidant activity of Bifidobacterium animalis 01 was investigated. Culture supernatant, intact cells, and intracellular cell-free extracts of B. animalis 01 were involved in this study. The antioxidant assays in vitro included lipid peroxidation assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, hydroxyl radical ((OH)-O-center dot) assay and superoxide anion (O-2(-)) assay. The antioxidant assays in vivo were conducted using mice model. Activities of antioxidative enzymes, malondialdehyde (MDA) content in serums and livers of aging mice were evaluated. Monoamine oxidase (MAO) activity and lipofuscin level in brains of aging mice were also characterized. Results showed that culture supernatant, intact cells and intracellular cell-free extracts of B. animalis 01 could effectively scavenge free radicals, significantly enhance mice's activities of antioxidative enzymes and reduce mice's MDA content, lipofuscin level and MAO activity. Our results indicated that B. animalis 01 has the potential to be developed into a dietary antioxidant supplements.