In this work an automatic flow procedure for L-alanine determination in the synthesis medium without a prior sample treatment is described. The method was based on oxidation of L-alanine by L-amino acid oxidize followed by the reaction of the H2O2 with luminol and chemiluminescence detection. In order to overcome the severe alkaline condition of the synthesis medium, a zone sampling strategy was employed for automatic sample dilution and pH adjustment. The procedure allows L-alanine determination in the range of 0.5 to 25.0 mmol L-1, a limit of detection of 0.08 mmol L-1 and a variation coefficient lower than 4% (n = 10). Other advantageous feature such as a sampling throughput of 44 determination per hour and low reagent consumption, 0.2 mg luminol and 16.4 mg hexacinoferrate(III) per determination, respectively, were also achieved.