Ovalbumin (OVA), a major allergen in egg -induced food allergy, is widely used in the food industry. This study investigated the structural and antigenic changes of OVA after covalent conjugation with rosmarinic acid (RA). The SDS-PAGE and RA binding equivalent analysis confirmed that both the radical and alkaline methods successfully prepared the OVA -RA conjugates. LC-MS/MS assay identified 20 and 15 modified sites of RA on OVA in the radical and alkaline OVA -RA conjugates, respectively. The structural changes of OVA were indicated by fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), and ultraviolet absorption (UV) spectrometry. Following conjugation with RA, the antioxidant capacities of OVA for scavenging DPPH center dot and ABTS center dot+ showed significant enhancement. Subsequently, the impact of RA on the antigenicity of OVA was assessed both in vitro and in vivo. The ELISA analysis revealed a notable reduction in the binding capacity of OVA to IgG and IgE after covalent conjugation with RA. Additionally, the passive cutaneous anaphylaxis (PCA) assay demonstrated lower allergenicity in radical and alkaline OVA -RA conjugates compared to native OVA. These findings collectively suggest that OVA -RA conjugates hold promise for the development of hypoallergenic food.