Abstract: A1 β-casein and A2 β-casein produce different substances after digestion due to differences in primary structure. Exploring the biological functions of A1/A2 β-casein was further realized by analyzing and monitoring the changes in this protein during digestion to explore its possible bioactive effects on the human body. In this study, allele-specific PCR was initially performed on milk samples collected from different cows in the pasture to identify A1 and A2 milk types, revealing that the predominant genotype was for A2 milk. Afterwards, β-casein isolated from A1 and A2 milk was purified and characterized with the help of anion exchange chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Finally, a static in vitro digestion model was constructed to compare A1 and A2 β-casein in terms of digestive properties. The results showed that the concentrations of isolated and purified A1 and A2 β-casein were 0.62 mg/mL and 0.66 mg/mL and the purities were 95.28% and 96.60%. After 5.0 h of static in vitro simulated digestion, the final digestibility of both β-caseins was around 90%, and the difference was not significant. A2 β-casein exhibited a higher degree of hydrolysis of 25.34% from the beginning of the intestinal digestion phase until the end of digestion. With the increase of gastrointestinal digestion time, the particle size of the two β-caseins decreased and the absolute value of the potential increased, and the degree of change in particle size and potential was greater for A2 β-casein than for A1 β-casein. In conclusion, the present study revealed the differences in the physicochemical properties of A1 and A2 β-caseins during static in vitro digestion and indicated to some extent that A2 β-caseins may have better digestive properties than A1 β-caseins, which provides a certain scientific basis for further expanding the application of A2 β-caseins in dairy products.