High hydrostatic pressure (HHP) is extensively utilized in the field of food processing due to its remarkable ability to preserve the freshness of food. The potential antigenicity of β-lactoglobulin (β-LG) in whey protein isolate (WPI, 3%) treated by HHP was detected by enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies. Furthermore, the impact of pressure-induced structural alterations on the emulsification properties and antioxidant activity of WPI was investigated. The findings revealed that pressures exceeding 300 MPa resulted in molecular aggregation, the formation of inter-molecular disulfide bonds, and an increase in surface hydrophobicity (H0). The percentage of β-sheet decreased along with the pressure. The results showed the increment of α-helix and β-turn with pressure. ELISA demonstrated a significant reduction in the antigenicity of β-LG following HHP treatment (100–600 MPa), with a slight recovery observed at 300 MPa. These spatial structural modifications led to the unfolding of the β-LG molecule, thereby enhancing its digestibility. Moreover, HHP treatment substantially improved the antioxidant properties, with the exposure to hydrophobic amino acids contributing to increased antioxidant properties and emulsion stability.