A novel and simple method for the selective cleanup and preconcentration of fluoroquinolone antibiotics in environmental water samples has been developed using molecularly imprinted polymer solid-phase extraction (MISPE). The molecularly imprinted polymer (MIP) has been prepared using enrofloxacin (ENR) as the template and a stoichiometric quantity of urea-based functional monomer to target the single oxyanionic moieties in the template molecule. The selectivity of the material for enrofloxacin, and structurally related and non-related compounds, has been evaluated using it as stationary phase in liquid chromatography. The novel polymer and the corresponding non-imprinted material (NIP) have been characterised using nitrogen adsorption-desorption isotherms and scanning electron microscopy. Various parameters affecting the extraction efficiency of the materials in the MISPE procedure were evaluated in order to achieve optimal preconcentration and to reduce non-specific interactions. The optimized MISPE/HPLC with fluorescence detection (FLD) method allows direct extraction of the antibiotics from the aqueous samples followed by a selective washing with acetonitrile/water (0.1M 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer, pH 7.5) (10/90, v/v) and elution with 2% trifluoracetic acid in methanol. Good recoveries and precision, ranging between 66 and 100% (RSD: 2-12%, n=3) for danofloxacin, enrofloxacin, oxolinic acid and flumequine, and moderate recoveries (15-40%, RSD 4-9%, n=3) for norfloxacin, ciprofloxacin, lomefloxacin and sarafloxacin, have been obtained for river water samples fortified with 0.50, 0.75 and 1.0microgL(-1) of all the antibiotics. The method detection limits ranged between 0.01 and 0.30microgL(-1) for all the antibiotics tested, when 100mL water samples were processed. The results demonstrate the applicability of the optimized method for the selective extraction of fluoroquinolones in environmental water samples at the ngL(-1) level.