Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) were used to investigate the conformational changes of heated whey protein (WP) and the corresponding changes in the hydrolysates immunoreactivity were determined by competitive enzyme-linked immunosorbent assay (ELISA). Results showed that the contents of α- helix and β-sheet of WP did not decrease much under mild heating conditions and the antigenicity was relatively high; when the heating intensity increased (70 ℃ for 25 min or 75 ℃ for 20 min), the content of α- helix and β-sheet decreased to the minimum, so was the antigenicity; However, when the WP was heated at even higher temperature and for a longer time, the β-sheet associated with protein aggregation begun to increase and the antigenicity increased correspondingly. It was concluded that the conformations of heated WP and the antigenicity of its hydrolysates are related and the optimum structure for decreasing the hydrolysates antigeniity is the least content of α-helix and β-sheet. Establishing the relationship between the WP secondary structure and WP hydrolysates antigenicity is significant to supply the reference for antigenicity reduction by enzymolysis.