The results in this study demonstrated that flavonoids rich extract from R. tomentosa berries demonstrated remarkable antioxidant activities on the DPPH radical scavenging activity, reducing power and inhibition of lipid peroxidation activity, as well as high in superoxide anion radical and hydroxyl radical scavenging activities. The flavonoids rich extract also increased the activities of SOD and GSH-Px activities, and decreased the MDA content in serum of mice. Based on the study in vitro and in vivo, it is predicted that the flavonoids rich extract prepared in this study can be a potential resource of natural antioxidants to be used in functional foods and health products. Furthermore, based on the results of UPLC–TOF-MS/MS, six known flavonoid compounds having significant biological activities were putatively identified. R. tomentosa berries should be used as a new natural resource to prepare these health-promoting flavonoid compounds. However, some other unknown flavonoid compounds in R. tomentosa berries were not yet identified in this study, and the kind of flavonoids playing a dominant role in the antioxidant activities was not clear. Thus, further fractionation and identification of flavonoids are in progress in our team. Moreover, other constituents in R. tomentosa berries and their biological activities will be investigated in future studies.