In this study, a high pressure liquid chromatography method with fluorescent detector was developed to analyze blood galactose, lactose and glucose simultaneously. Plasma sugars were prepared as fluorescent derivatives to react with FMOC-hydrazine (9-fluorenyl methyl chloroformate). A C18 reversed phase column and a fluorescent detector were used and run in ambient. The resolution index of galactose and glucose derivatives in the analytical method was 1.15. The coefficients of variation of the analysis were less than 7.5%. The concentration of FMOC-hydrazine did not significantly influence the analytical results for determination of the concentration of galactose. However, the ratios of acetonitrile in the mobile phase significantly affected the analysis of the fluorescent derivatives of sugars. The sensitivity of this method for galactose detection was 5 micrograms ml-1, and the required plasma volume for testing was only 25 microliters each. This analytical method was successfully applied to study the pharmacokinetics of galactose in vivo in a rabbit model.