To explore the antioxidant capacity of citrus flavonoids under different evaluation systems, chemical and biological methods were engaged to determine the antioxidant abilities of flavanones and polymethoxyflavones. Results showed that flavanones exhibited good antioxidant activity, while polymethoxyflavones had a weak ability to scavenge free radicals. Both flavanones and polymethoxyflavones exerted the ability to inhibit HO-induced oxidative stress, but the effective concentration of polymethoxyflavones was lower. Further exploration showed that neohesperidin and tangeretin selectively regulated antioxidant enzyme activity, both in vitro and in vivo. Tangeretin also maintained the expression of antioxidant enzymes in L02 cells and in ICR mice liver. The mechanism exploration showed that both neohesperidin and tangeretin promoted the expression of NRF2 and inhibit the expression of KEAP1, but tangeretin could inhibit the ubiquitination of NRF2 by inhibiting CUL3. The mechanism was verified by CUL3 gene silencing. This study demonstrates a novel antioxidant mechanism of natural products.