A sensitive fluorescence (FL) technique is proposed for the determination of levofloxacin (LVX). The method is based on the fact that the weak FL signal of the Tb(III)-LVX system is strongly enhanced in the presence of gold nanoparticles. Gold nanoparticles were prepared by the citrate reduction of HAuCl(4) and characterized by transmission electron microscopy (TEM). Levofloxacin and Tb(III) ion form a fluorescence complex in aqueous solution, and its maximum emission wavelength was found at 545 nm. Optimal conditions for the formation of the levofloxacin-Tb(III) complexes were studied. Levofloxacin was detected by measuring the FL intensity, which increases linearly with the concentration of LVX in the range 6.2 × 10(-10)-2.6 × 10(-8) mol/L. Recovery of the target analytes was > 96% with good quality parameters: linearity (r(2) > 0.996), limit of detection (LOD) and limit of quantification (LOQ) values 2.1 × 10(-10) mol/L and 7.2 × 10(-10) mol/L, and run-to-run and day-to-day precisions with relative standard deviations (RSDs) around 3%. Thus, the proposed method can be successfully applied to the routine determination of levofloxacin in pharmaceutical preparations.