Purpose: As a typical Chinese herbal medicine, Radix Glycyrrhizae (RG) possesses various pharmacological effects involved in antioxidant ability. However, its antioxidant has not been explored so far. The aim of the study was to investigate its antioxidant ability, then further discuss the antioxidant mechanism. Methods: RG was extracted by ethanol to obtain ethanolic extract of Radix Glycyrrhizae (ERG). ERG was then determined by various antioxidant methods, including DNA damage assay, DPPH assay, ABTS assay, Fe3+-reducing assay and Cu2+-reducing assay. Finally, the contents of total phenolics and total flavonoids were analyzed by spectrophotometric methods. Results: Our results revealed that ERG could effectively protect against hydroxyl-induced DNA damage (IC50 517.28±26.61μg/mL). In addition, ERG could scavenge DPPH· radical (IC50165.18±6.48μg/mL) and ABTS+ radical (IC507.46±0.07μg/mL), reduce Fe3+ (IC50 97.23±2.88 μg/mL) and Cu2+ (IC50 59.21±0.18 μg/mL). Chemical analysis demonstrated that the contents of total phenolics and flavonoids in ERG were 111.48±0.88 and 218.26±8.57 mg quercetin/g, respectively. Conclusion: Radix Glycyrrhizae can effectively protect against hydroxyl-induced DNA damage. One mechanism of protective effect may be radical-scavenging which is via donating hydrogen atom (H·), donating electron (e). Its antioxidant ability can be mainly attributed to the flavonoids or total phenolics.