Abstract: In order to quantify the content of lactulose in liquid milk easily and accurately,the HPLC method for determination of lactulose in liquid milk was improved. The method used stable modified amide-based chromatographic column for separation,and eluted with acetonitrile-water mobile phase for 40 minutes gradiently. The results indicated that when the injection volume was 3 μL,the resolution was 1.5,which met the chromatographic separation detection. The optimized method has been verified that lactulose has a good linear relationship in the range of 50~2500 mg/kg,(R²=0.9997),the detection limit and the quantitative limit of lactulose were 15 mg/kg and 50 mg/kg respectively. The spiked recoveries of lactulose were in the range of 90.3%~105.6%,and relative standard deviations(n=6)in the range of 3.4%~3.9%. The proposed method was used to detect 27 actual milk samples,and the results were within a reasonable range. This method was economical and accurate for the daily detection of lactulose in liquid milk.