This study investigated the interactions between Capsaicinoids (CAPs) and beef myofibrillar proteins (MPs) in a peroxyl radical system and elucidated the antioxidant mechanisms of CAPs by multispectral and molecular docking. Results showed that low concentration CAPs prevented the oxidative changes of protein structure caused by the attack of AAPH radicals on MPs, while high concentration of CAPs changed the structure of the proteins to form more small molecule aggregates, and reduce the binding of actin-myosin, which was conducive to the tenderization of the meats. CAPs bound to the MPs through hydrophobic interaction, hydrogen bonding and electrostatic interaction, altering the secondary and tertiary structure of MPs, increasing the α-helix content of MPs, and improving the antioxidant structural stability of MPs. This study can provide a theoretical basis for the utilization of CAPs in prefabrication meat processing, and provide a theoretical support for protein antioxidant strategies in spicy dishes.