Aflatoxin B₁ （AFB₁） is widely distributed and highly hepatotoxic, which can cause oxidative damage to human liver. In this study, a rat model of acute liver injury was established by injecting AFB₁ to explore the intervention effect and method of chitosan oligosaccharide （COS） with antioxidant activity on liver injury. The experiment was designed with blank group, COS control group, model group, COS intervention group, and positive control group. Consecutive gavage for 8 days, the model group, COS intervention and positive control group were injected with 1 mg/kg AFB₁ only on the 6th day. The intervention effect of COS on liver injury was evaluated by serum liver function indexes, lipid peroxide content, antioxidant enzyme activity and liver tissue sections in rats, and differentially expressed genes （DEGs） were obtained by RNA sequencing （RNA-Seq） technology to analyse the intervention mode of COS. The results showed that compared with the model group, the COS intervention group significantly decreased the serum liver function indexes and lipid peroxide content, and increased the activity of antioxidant enzymes； the observation of liver slices showed that COS was beneficial to improve the liver morphology of rats. RNA-Seq data showed a total of 968 DEGs in the COS intervention group compared with the model group, and the enriched gene ontology （GO） entries were mainly related to liver metabolic function, oxidative stress response and protein synthesis. In conclusion, COS has a certain intervention effect on AFB₁-induced acute liver injury, which provides a theoretical basis for COS to prevent AFB₁-induced liver injury.