Warfarin (3-(α-acetonylbenzyl)-4-hydroxycoumarin) is a widely used anticoagulant rodenticide which has strong toxic effects in humans and animals. A new multisyringe flow injection analysis (MSFIA) set-up for the spectrofluorimetric determination of warfarin in waters at trace levels is presented. Preconcentration on a solid-phase (octadecyl chemically-bonded silica-gel-based beads) without prior derivatization, and elution with dimethyl sulfoxide (DMSO) containing monomeric units of the cetyltrimethylammonium chloride (HTAC) surfactant resulted in a quantification limit (50 ng l −1) lower than those reported to date, reaching the levels demanded by regulatory authorities in drinking waters. Several variables such as eluent composition, sample and eluent volumes, retention and elution flow rates and sample pH were studied in detail. A mass calibration may be used to determine warfarin in the wide range from 50 ng l −1 to 64 μg l −1 for sample volumes between 0.2 and 12 ml. An enrichment factor of 155, a sample throughput of 12 h −1 and a repeatability better than 2% were achieved by preconcentration of 16 ng of the target compound from 2 ml of sample.