A rapid precolumn derivatization combined with high performance liquid chromatography (HPLC) with fluorescence detection method for the determination of the 1-deoxynojirimycin (DNJ) content in functional milk powder. Samples were extracted with 60% methanol in water. In pH 8.5 borate buffer solution, o-phthalaldehyde was added as a derivatizing agent to remove the interference from primary amines. DNJ in samples reacted with 9-fluorenylmethoxycarbonyl chloride to form a complex with fluorescence absorption. The separation was performed on a C₁₈ column (150 mm × 4.6 mm, 2.7 μm). Mobile phase A was 10 mmol/L sodium acetate buffer solution, and mobile phase B was acetonitrile. Gradient elution was performed at a flow rate of 1.5 mL/min. The column temperature was 45 ℃. The excitation wavelength was 275 nm. The emission wavelength was 320 nm. Good separation of DNJ from other components was achieved. The detection limit was 4 ng/mL, and the calibration curve was linear in the range of 0.01 to 50 μg/mL, with a determination coefficient (R²) > 0.999. The recovery rates ranged from 101.8% to 104.1%. The method was validated to be stable and reliable, and could be used as an effective method for determining the content of DNJ in milk powder.