The thermal denaturation of β-lactoglobulin of milk was investigated by using DSC, circular dichroism spectra, and fluorescence spectra systematically. And its corresponding immunogenicity was tested with ELISA experiments. In addition, bioinformatics methods were also used to explain the spectral characteristics and its corresponding immunogenic change, analyze the conformational changes of the protein during thermal denaturation, and discuss the relationship between conformational change of β-lactoglobulin caused by heat and immunogenic change. Results from this study should be useful to the establishment of a spectral method examining the extent of thermal denaturation of the food allergen, and be helpful to the understanding of the mechanism of immunogenic change of food allergen treated by heat.