Diethyl phthalate (DEP) is a commonly used phthalate ester (PAE) plasticizer known for its excellent plasticity and flexibility. Environmental exposure to DEP may have adverse effects on human health. Currently, few studies have focused on the specific effects and mechanisms on aquatic organisms. This study investigated the effects of oxidative stress effects and genotoxicity of DEP on zebrafish liver, as well as molecular interactions with antioxidant enzymes both in vitro and in vivo. The results revealed that exposure to DEP led to changes in the activity of antioxidant enzymes, which may be due to changes in the structure and conformation of antioxidant enzymes induced by DEP. This disruption of redox homeostasis in the liver of adult zebrafish led to oxidative stress, resulting in oxidative damage and genotoxicity in tissues. These damages did not exhibit concentration or time dependence, as indicated by integrated biomarker response (IBR) analysis for zebrafish. This study established a rapid and effective ecological risk assessment model to evaluate the biological toxicity of DEP through animal and molecular experiments, which could provide technical support for the risk management of DEP and the formulation of scientific and rational management strategies. Additionally, it may serve as a scientific reference for preventing and treating diseases caused by DEP.