Oxidative stress is highly associated with the development of cardiovascular diseases and cancer and has drawn great attention. Natural products suggest a potential role in prevention of these disorders. The aim of this study was to investigate the antioxidant and anti-cancer properties of Chinese bayberry leaves (Myrica rubra Sieb. et Zucc.) flavonoids (BLF) comprehensively through the combination of in vitro digestion, chemical and cellular antioxidant assays. Based on the LC/MS data, the major flavonoids of BLF were myricitrin and quercetin 3-rhamnoside. BLF owned strong chemical and cellular antioxidant activity (CAA) with its CAA value at 4253.884 ± 435.366 μmol of QE/100 g DW. After the in vitro digestion, the total flavonoids content, myricitrin and quercetin 3-rhamnoside decreased significantly (P < 0.05). Lower levels of the total flavonoid content and cellular uptake of myricitrin and quercetin 3-rhamnoside might contribute to the lower CAA value of digested BLF (DBLF). However, DBLF still owns considerable chemical antioxidant activities and CAA compared with many plants. Furthermore, both BLF and DBLF exhibited dose-dependent relationship against HepG2 proliferation. Taken together, BLF has a great potential to be developed as a natural antioxidant for promoting public health.