The aim of this study was to compare the sensitization of αS1-CN in cow and goat's milk in a mouse model. Fifty mice were divided into control group, adjuvant control group, cow's milk αS1-CN sensitized group, goat's milk αS1-CN sensitized group and cross sensitized group. Cow's and goat's milk αS1-CN were used to establish a mouse sensitization model. The results showed that cow's milk αS1-CN had higher allergenicity than goat's milk αS1-CN, as can be seen in significantly increased s-IgE and Th2 cell-related inflammatory factors, the proportion of Th2, and the expression of Th2 cell-related transcription factors. Furthermore, the sensitization of cow's milk αS1-CN damaged the intestinal barrier of mice, caused the leakage of LPS, activated the TLR4-NFκB pathway, and thus resulted in the increase of IFN-γ. In addition, mice allergic to cow's milk αS1-CN were less sensitized to goat's milk αS1-CN.