Flavonoids are famous for their diverse sources, strong biological activity, and low toxicity and could be used as a natural antioxidant in animal husbandry. In this study, the purification process and antioxidant activity of flavonoids from fermented dandelion were investigated. The adsorption and desorption characterizations of AB-8 macroporous resin for flavonoids from fermented dandelion (FD) were determined and purification parameters were optimized. Qualitative analysis using UPLC-MS/MS analysis was explored to identify the components of the purified flavonoids of FD (PFDF). The antioxidant activity of PFDF in vitro and in vivo was analyzed. The optimum purification parameters were as follows: a sample concentration of 2 mg/mL, 120 mL of the sample volume, a pH of 2.0, and eluted with 90 mL of 70% ethanol (pH 5). After purification, the concentration of the flavonoids in PFDF was 356.08 mg/mL. By comparison with reference standards or the literature data, 135 kinds of flavonoids in PFDF were identified. Furthermore, PFDF had a strong reducing power and scavenging ability against 8-hydroxy radical and DPPH radical. PFDF can effectively reduce the oxidative stress of zebrafish embryos and IPCE-J2 cells by modulating antioxidant enzyme activities. In summary, the purified flavonoids from fermented dandelion have good antioxidant activity and display superior potential as a natural antioxidant in animal husbandry.