This paper describes a method for determination of vitamin K2 (menaquinone-7, MK-7) in milk powder using high performance liquid chromatography (HPLC). The sample was hydrolyzed with lipase (250 mg/g) for 2 h, saponified with potassium carbonate (500 mg/g), extracted with n-hexane, concentrated by rotary evaporation, reconstituted with methanol/isopropanol (4:1), and post-derivatized with C18 column and reduced column before being analyzed by HPLC with a fluorescence detector. The analyte was quantified by external standard method. The results showed that the peak of MK-7 appeared at about 19.47 min, and the calibration curve for it showed a good linear relationship in the range of 0.0–1.5 μg/mL (R2 = 0.999 95). The recoveries for blank samples spiked at 5, 10 and 25 μg/100 g were between 87.9% and 94.3%, with relative standard deviations of 0.9%–6.4%. When 2 g of sample was taken and the volume was made up to 1 mL, the detection limit was 0.15 μg/100 g and the quantification limit was 0.5 μg/100 g. The method was simple with high accuracy, sensitivity and stability, and could be used for the detection of vitamin K2 (MK-7) content in milk powder.