An innovative automatic purge-and-trap (P&T) system coupled with fluorimetric sequential injection (SI), for the on-line separation and preconcentration of volatile compounds, is presented. The truth of concept is demonstrated for the ammonium fluorimetric determination in environmental water samples with complex matrices without any pretreatment. The P&T flow system comprises a thermostated purge-vessel where ammonium is converted into gaseous ammonia and a trap-vessel for ammonia collection. This configuration results in matrix removal as well as analyte preconcentration, avoiding membrane-associated problems. All the main parameters affecting the efficiency of a P&T system were studied and optimized. The proposed method is characterized by a working range of 2.7–150.0 μg L−1 of NH4+, with a detection and quantification limit of 0.80 and 2.66 μg L−1, respectively, for a 10-mL sample consumption. The accuracy of the method was assessed by recovery assays in seawater, estuarine, and lake water samples as well as by the analysis of standard reference material.