β-Casein, an important protein found in bovine milk, has significant potential for application in the food, pharmaceutical, and other related industries. This review first introduces the composition, structure, and functional properties of β-casein. It then reviews the techniques for isolating β-casein. Chemical and enzymatic isolation methods result in inactivity of β-casein and other components in the milk, and it is difficult to control the production conditions, limiting the utilization range of products. Physical technology not only achieves high product purity and activity but also effectively preserves the biological activity of the components. The isolated β-casein needs to be utilized effectively and efficiently for various purity products in order to achieve optimal targeted application. Bovine β-casein, which has a purity higher than or close to that of breast β-casein, can be used in infant formulas. This is achieved by modifying its structure through dephosphorylation, resulting in a formula that closely mimics the composition of breast milk. Bovine β-casein, which is lower in purity than breast β-casein, can be maximized for the preparation of functional peptides and for use as natural carriers. The remaining byproducts can be utilized as food ingredients, emulsifiers, and carriers for encapsulating and delivering active substances. Thus, realizing the intensive processing and utilization of bovine β-casein isolation. This review can promote the industrial production process of β-casein, which is beneficial for the sustainable development of β-casein as a food and material. It also provides valuable insights for the development of other active substances in milk.