Based on the existing detection method data,this detection method improved the separation effect of A1β- Casein and A2β- casein by changing the column temperature and gradient elution conditions,so as to better analyze the content of A1β- Casein and A2β- casein in dairy products. In this assay,guanidine hydrochloride and dithiothreitol were used to denature β-caseinthe selection of mobile phase B（acetonitrile）,A（0.1 % TFA aqueous solution）,gradient elution,flow rate 0. 5 mL/min,reversed-phase liquid chromatography column,column temperature 50 ℃,214 nm detection.The method can effectively separate A1β-casein and A2β-casein,and the peak time was stable. A1β-casein in 0.16~1.65 mg/mL concentration range showed a good linear relationship,regression equation y=1.25×106x-1.44x105,R2=0.9985,R=0.9992,the RSD of precision was less than 2.0%,the range of recovery rate were 96.4%~105.0%.A2 β-casein in 0.34~3.35 mg/mL concentration range showed a good linear relationship,regression equation y=3.16×106x-3.16×105,R2=0.9988,R=0.9994,the RSD of precision was less than 2.0%,the range of recovery rate were 97.5 %~105.0 %. The results showed that the method was simple,reproducible and accurate. It can be used as a technical reference for further research and application of A1β-casein and A2β-casein in dairy products.