A sensitive chemiluminescence method coupled with flow-injection has been proposed for the determination of gatifloxacin based on the reaction with luminol and hydrogen peroxide catalyzed by silver nanoparticles (Ag NPs). Therefore, the application of luminol-H2O2-Ag NPs system was exploited for the determination of gatifloxacin in the linear range of 1.2 × 10^(-10) - 4.4 × 10^(-8) g mL^(-1) with a detection limit of 1.17 × 10^(-11) g mL^(-1). The proposed method offers high sensitivity, wide linear range and good stability which can be successfully applied to the determination of gatifloxacin in pharmaceutical preparations and human urine.