The effect of heat treatment on the allergenicity and microstructure of ovomucoid was studied by enzyme-linked immunosorbent assay (ELISA), CD spectra, ANS fluorescence probe emission spectra and UV absorption spectra. The results showed that the allergenicity of ovomucoid decreased with increasing temperatures and time. CD spectra were used to examine the changes in the secondary structure of the ovomucoid treated by different temperatures. When treated by different temperatures, the mutual transformation between a-helix, b-sheet, b-turn and the random coil was observed, and the disorderliness of the secondary structure was increased with α-helix and β-sheet decreasing, but the random coil increased correspondingly. The ANS fluorescence probe emission spectra analysis demonstrated that heat treatment induced reducing in surface hydrophobicity with the increase in heat temperatures. With the increase in heat temperatures, the amino acid residues with the UV-absorption were exposed gradually, and the UV-absorption maximum value increased. The results indicated that when treated by heating, the changes in the microstructure of ovomucoid can lead to the changes in the allergenicity of ovomucoid.