Zearalenone (ZEN), a mycotoxin present in cereals, poses significant health risks to animals and humans due to its estrogenic effects. Numerous studies on the enzymatic detoxification of ZEN have predominantly focused on reducing the parent toxin to assess the enzyme’s efficacy, yet there is limited research on the identification and toxicity evaluation of the enzymatic degradation products. This study investigated the enzymatic degradation mechanisms of ZEN using commercial peroxidase (POD) and laccase (LC), with a focus on identifying degradation products and assessing their hepatotoxicity effects. Molecular docking and dynamics simulations elucidated the binding mechanisms between these enzymes and ZEN, revealing strong interactions that facilitate efficient detoxification. Subsequent analysis employing ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) successfully identified crucial degradation products. Hepatic toxicity of the enzymatic degradation products was comprehensively assessed in HepaRG liver cells through systematic measurements of cell viability, oxidative stress, apoptosis, mitochondrial membrane potential, and molecular metabolic profiles. Our findings demonstrate that both POD and LC exhibit significant efficacy in mitigating hepatocyte toxicity induced by ZEN, thereby highlighting their potential utility in enhancing food safety. This research provides essential data for safety evaluation regarding enzymatic detoxification of ZEN while offering theoretical and technical resources for risk assessment related to mycotoxin enzymatic detoxification.