A pre-column derivatization liquid chromatographic method was developed for the simultaneous determination of both glucosamine and galactosamine in selected food samples. The derivatization of glucosamine and galactosamine with 9-fluorenylmethoxycarbonyl chloride was accomplished under alkaline conditions and UV irradiation. When the detection wavelength was 265 nm and acetonitrile-0.1% phosphoric acid was the mobile phase at a flow rate of 1.0 mL/min, the linear range of ultraviolet intensity of glucosamine and galactosamine was 10–100 μg/mL,correlation coefficient of glucosamine was 0.9994 and correlation coefficient of galactosamine was 0.9993. The standard recovery of three kinds of food was in the range of 94.09–114.5%, the relative standard deviation (RSD) was 0.38–5.29%, the detection limit was 1.0 mg/kg, and the quantitative limit was 4.0 mg/kg. The results of this research indicate that this method is suitable for the simultaneous determination of glucosamine and galactosamine in food.