The main purpose of this study was to develop an inexpensive, simple, rapid and sensitive chemiluminescence (CL) method for the determination of glutamine (Gln) using a flow-injection (FI) system. Gin was found to strongly inhibit the CL signal of the luminol-H2O2-CuSO4 system in Na2B4O7 solution. A new FI-CL method was developed for the determination of Gin. Parameters affecting the reproducibility and CL detection were optimized systematically. Under the optimized conditions, the corresponding linear regression equation was established over the range of 5.0 x 10(-7) to 2.5 x 10(-6) mol/L with the detection limit of 1.8 x 10(-8) mol/L. The relative standard deviation was found to be 1.8% for 11 replicate determinations of 1.5 x 10(-6) mol/L Gln. The proposed method has been satisfactorily applied for the determination of Gln in real samples (Marzulene-s granules) with recoveries in the range of 98.7-108.6%. The minimum sampling rate was about 100 samples/h. The possible mechanism of this inhibitory CL was studied by fluorescence spectrophotometer and UV-vis spectrophotometer. Copyright (C) 2009 John Wiley & Sons, Ltd.