An immunoaffinity column purification-high performance liquid chromatographic method was developed for the determination of vitamin B12 in milk powder.The sample was dissolved with phosphate buffer,and purified through the vitamin B12 immunity affinity column.After eluted with methanol,and concentrated,the target compound was directly measured on a Symmstry C18 column.The optimal conditions were as follows:column temperature:35 ℃,mobile phase:0.03 mol/L potassium dihydrogen phosphate（phosphate adjustable pH 3.0）-acetonitrile（85∶15）,flow rate:1.0 mL/min,detection wavelength:361 nm. Under the optimal conditions,the calibration curve was linear in the range of 20-300 μg/L ,with correlation coefficient（r） of 0.999 7 and a detection limit of 0.6 μg/kg.The spiked recoveries were in the range of 90%-96% with RSD of 1.1%-2.4%.Compared with the national standard method,the proposed method has the advantages of easy sample handling,high sensitivity,good reproducibility and short analysis time, and could meet the requirement for the determination of trace amounts of vitamin B12 in milk powder and infant formula.