Antioxidant activity of enzymatic hydrolysate of yak casein was investigated by phenanthroline-Fe2+ method.By univariate analysis,effects of the reagent dosage and reaction condition on absorbance and scavenging rate of hydroxyl free radicals were investigated.The optimal reaction conditions were as followes: 1 mL of 1.5 mmol/L Phenanthroline,2 mL of pH7.4 phosphate buffer and 1mL antioxidants was successively collected in a test tube with stopper and mixed fully,then 1 mL of 0.5 mmol/L ferrous sulfate solution was added and mixed,later 1mL of 0.1% hydrogen peroxide was added.Absorbance of sample was detected at 536 nm using ultraviolet spectrophotometer after reaction at 38℃ for 50 min.The results showed that improved method is reproducible to results of determination and applicable to determination of antioxidant activity of hydrolysate of casein.