A new and sensitive application of chemiluminescence detection has been developed for the determination of the pesticide thiacloprid in water. It was based on the on-line photoreaction of thiacloprid in a basic medium, with quinine acting as the sensitizer of the chemiluminescent response; cerium (IV) in sulfuric acid medium was used as the oxidant. High degrees of automation and reproducibility were achieved using a flow-injection analysis (FIA) manifold. The validation of the method was performed in terms of selectivity, linearity, limit of detection (LOD), precision, and accuracy. Liquid chromatography with ultraviolet (UV) detection was used as reference for mineral, tap, ground, and spring water samples. The proposed method is fast (with a throughput of 130 h<sup>−1</sup>), sensitive (LOD of 0.8 ng mL<sup>−1</sup> without preconcentration steps and of 0.08 ng mL<sup>−1</sup> with solid-phase extraction [SPE]), low cost, and possible to couple with separation methods for the simultaneous determination of other pesticides. The enhanced chemiluminescence intensity was linear with the thiacloprid concentration above the 2-80 and 80-800 ng mL<sup>−1</sup> ranges. A possible reaction mechanism is also discussed.

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