Abstract: The β-casein of bovine milk contains a variety of variants, among which A1-β-casein (A1) and A2-β-casein (A2) are the two most common variants. Because of only few differences between with A1 and A2 in amino acid sequence, it is difficult to preparation of A1 and A2 proteins with higher purity by conventional separation and purification methods. In this study, recombinant plasmid of pET28a（+）-CSN2-A1 and pET28a （+）-CSN2-A2, which contained the target genes of CSN2-A1 and CSN2-A2 were constructed by molecular biological methods, respectively. Then two recombinant vectors were introduced into Escherichia coli BL21 for induced expression and purification, respectively. The results showed that abundant of proteins could be obtained by induced expression at 0.2 mmol/L IPTG at 37 ℃ for 4 h. However, the results of SDS-PAGE showed that the target proteins were expressed in the form of inclusion bodies, and existed in the pellet after cell disruption. In addition, the purity of more than 90% (SDS-PAGE) of the A1 and A2 recombinant proteins were obtained by several processes, which contained dissolution, nickel affinity chromatography, renaturation and qualification. Thereby, it could provide a new way for the preparation of A1 and A2 proteins.