A sensitive fluorescence (FL) method has been proposed for the determination of prulifloxacin (PUFX) using Eu3+` as fluorescence probe. The method is based on the enhancement of the FL intensity of Eu3+-PUFX in the presence of silver nanoparticles (Ag NPs). The enhanced FL intensity of the Eu3+-PUFX-Ag NPs system at 614 nm showed a good linear relationship with the concentration of PUFX within the range of 2.8×10^(-8)-1.1×10^(-5) mol L^(-1) with correlation coefficient of 0.9998. The limit of detection was found to be 5.8×10^(-9) mol L^(-1). The proposed method has been successfully applied for the determination of PUFX in pharmaceutical samples and bio-fluids with high sensitivity and selectivity.