A method of high performance liquid chromatography(HPLC) with fluorescence detector(FLD) was developed for the simultaneous determination of 5 substances in the microbial enzymatic reaction system of L-alanyl-L-glutamine,including L-alanyl-L-glutamine,L-glutamine,L-alanine methyl ester,L-glutamic acid and L-alanyl-L-alanine.Standard substances and samples were derivated with phthaldialdehyde(OPA) by online automated precolumn derivatization.The analytes were separated on a C18 column using acetonitrile(12%) and 12.5 mmol/L potassium phosphate buffer(88%,pH 7.4) as mobile phase by isocratic elution and analyzed quantitatively by the external standard method at an excitation wavelength of 338 nm and an emission wavelength of 450 nm.The flow rate was set at 1 mL/min and the column temperature was 40 ℃.The calibration curves for five standard substances were all linear in a broad range with correlation coefficients more than 0.999 4.The average recoveries of five substances at three spiked levels ranged from 93.5% to 104.9% with relative standard deviations(RSDs) less than 7%.Injecting six times every two hours one day,the RSD values of retention time for five substances were all less than 0.12%,and the RSD values of peak area of five substances were all less than 1.2%.The method was simple,sensitive,accurate and repeatable with a high resolution.