In order to explore the mechanism of liver injury induced by florfenicol (FFC) in broilers, one hundred and twenty broilers were randomly divided into six groups, twenty broilers in each group. Except for control group, the other five groups were given different doses of FFC (0.15g/L, 0.3g/L, 0.6g/L, 1.2g/L and 1.8g/L) in drinking water. After five days of continuous use, blood was collected from the subpterional vein and the chickens' liver were obtained. Chicken weight gain and liver indices were calculated; blood routine analysis was performed; the oxidative stress and apoptosis of hepatocytes was detected. The results showed that compared with the control group, except for 0.15g/L FFC, the other doses of FFC significantly decreased the weight gain, white blood cell (WBC) and platelet (PLT) contents in blood, 0.3g/mL FFC and 1.8g/L FFC significantly reduced the content of hemoglobin (RGB) (P<0.05); all doses of FFC significant decreased red blood cell (RBC) increased Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) contents in serum of chickens (P<0.05), and significantly decreased the contents of albumin (ALB) and total protein (TP) in serum (P<0.05), but had no significant effect on alkaline phosphatase (ALP) contents(P>0.05). FFC significantly increased malondialdehyde (MDA) content in serum and liver tissues, but decreased glutathione (GSH), Superoxide dismutase (SOD) and catalase (CAT) content (P<0.05), and significantly inhibited the mRNA transcription and protein expression of antioxidant proteins nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone-1 (NQO-1)(P<0.05). FFC also inhibited the content and the transcription level of cytochrome P4501A1(CYP1A1) and CYP2H1 in liver (P<0.05). At the same time, FFC significantly promoted the apoptotic rate of hepatocytes and the mRNA transcription and protein expression of caspase-3 and caspase-6 (P<0.05). With the increase of FFC concentration, liver injury became more and more serious, which affected liver function in chickens by inhibiting enzyme activity in Nrf2-ARE pathway to increase oxidative stress and promoting apoptotic protein expression to accelerate hepatocyte apoptosis.