The fruits of Lycium barbarum are rich in flavonoids, which may contribute to the health-promoting function of Lycium barbarum. However, the composition of flavonoids in the fruits of Lycium barbarum (LBFs) has received little attention. Thus, the goal of this work was to identify more kinds of flavonoids from fruits of Lycium barbarum by liquid chromatography–mass spectrometry. The potential antioxidant and anti-inflammatory activities of LBFs in vitro were also investigated. Thirteen flavonoid compounds were identified in LBFs, of which daphnetin, 6,7-dihydroxycoumarin, astragalin, taxifolin, eriodictyol, naringenin, and chrysoeriol were identified for the first time in the fruits of Lycium barbarum, which greatly enriched the variety of flavonoids in the fruits of Lycium barbarum. LBFs showed a similar superior antioxidant activity to vitamin C. Furthermore, LBFs exhibited an anti-inflammatory activity by suppressing the production of nitric oxide and pro-inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1β, and interleukin-6, in lipopolysaccharide-treated RAW264.7 macrophage cells. This study demonstrated the potential development of LBFs as functional foods.