Abstract: A new HPLC method for analysis of β-carotene in milk powder was established.The retention time of β-carotene was about 3.5 min, phase were(n-hexane+cyclohexane+iso-propyl-alcohol=0.46+0.46+0.08)+ chloroform=99+1.Linear ranges of determination is 2.280～456.000 μg/mL,and the limits of determination of the analysis method is 0.220 μg/mL.Recovery founded in the milk powder sample is in the range of 90.2%～95.1%.