Abstract: To develop immunoaffinity column purification-high performance liquid chromatographic method for the determination of free folic acid in milk powder. Sample processing of the method for the sample dissolved with phosphate buffer, the sample liquid through folate immunoaffinity column purification, eluent of methanol, and concentrated to a direct determination after enrichment. These samples were separated on Waters C18 column (250mm×4.6mm, 5μm) , eluted with mixture of (20mg of sodium hexanesulfonate+10mL+1.3mL of glacial acetic acid and triethylamine the constant volume 1L, pH3.2) :methanol (80∶20, V/V) at 1mL/min, and detected at 280nm. Standard curve regression equationy=-185.68+290.29x, a correlation coefficient of R2=0.9997. Recovery was 88.2%, RSD was 1.99%. The method detection limit was 40μg/kg. Compared to the national standard method, the method had the advantage of easy sample handling, high sensitivity, good reproducibility and short analysis time to meet that modulation milk powder and infant formula milk powder folic acid content determination.