Abstract: Objective: To study the best purification method of Perilla flavone (PF), and to investigate its protective effect and potential mechanism on Paracetamol (APAP) - induced acute liver injury. Methods: Single factor experiments were used to determine the optimal conditions for the purification of PF by macroporous resin. BALB/c male mice were randomly divided into 6 groups, blank control group (CON), model group (MOD), diphenylene dibenzoate group (DDB), low dose group of PF (L-PF, 12.5 mg/kg), middle dose group of PF (M-PF, 25 mg/kg), and high dose group of PF (H-PF, 50 mg/kg). DDB and PF were given by continuous intragastric administration for 15 days according to the dosage. After the last administration for 1 h, APAP was injected intraperitoneally to establish liver injury model in each group except CON group. The serum levels of AST and ALT of mice were determined, and the levels of MDA, SOD, GSH, and GSH-PX in the liver of mice were tested. HE staining of the livers was performed to observe pathological changes. The expressions of Nrf2, Keap1 and HO-1 proteins in the livers were detected by Western Blot. Results: The optimal purification conditions were as follows: 10 g of D101 resin, 2.0 mg/mL of up-sampling solution, up-sampling volume of 30 mL, 50 mL of 80% ethanol solution elution. After purification, the flavonoid content was 70.32%±1.49%. Compared with CON group, the expressions of AST and ALT in the serum were significantly higher in MOD group (P<0.01), indicating successful modeling of liver injury. Compared with MOD group, the liver index, AST, ALT, MDA, GSH levels and Keap 1 protein expression in PF-given groups were significantly reduced by 25%~60% (P<0.01 or P<0.05), the concentrations of SOD, GSH-PX, and the expression levels of Nrf2 and HO-1 protein were significantly increased by 15%~35% (P<0.01 or P<0.05). HE staining results showed that PF could significantly improve APAP-induced liver injury. Conclusion: The PF can protect APAP-induced acute liver injury, and its mechanism is related to the Nrf2/HO-1 signaling pathway which regulates oxidative stress.