A new, simple, rapid and sensitive batch chemiluminescence (CL) method for determination of six fluoroquinolones (ciprofloxacin, gatifloxacin, levofloxacin, lomefloxacin HCl, ofloxacin and sparfloxacin) is proposd. The method is based on the CL generated during the oxidation of luminol by N-bromosuccinimide (NBS) in alkaline medium. The determination of the studied drugs is based on their inhibiting effect on the emission intensity of NBS-luminol chemiluminescent reaction. The effect of analytical variables on this CL system is discussed. The study was validated according to ICH guidelines. Under the optimum experimental conditions, the linear range is 50 to 400 ng/ml for ciprofloxacin, 50 to 600 ng/ml for gatifloxacin, lomefloxacin HCl and sparfloxacin and 25 to 400 ng/ml for levofloxacin and ofloxacin and the detection and quantitation limits for the studied drugs were not more than 4.88 and 14.80 ng/ml, respectively. The proposed method has been applied to detect the studied drugs in their pure forms and in different pharmaceutical formulations. The possible mechanism of the CL reaction was discussed.