In this study, a simple, rapid and accurate spectrophotometric method was proposed for the determination of the Levofloxacin using Nickel Oxide nanoparticles NiONPs (which were synthesized in a green way using dried mint leaves extract), It gave the highest value of absorption at 294 nm wavelength , and the linear relationship followed the Beer-Lambert law in the range of concentrations (2-24 µg/mL) with a good correlation coefficient, where its value was R = 0.9996 which indicates good linearity, the value of the molar absorptivity =2.9704×104 L/mol.cm , and the value of the Sandell’s significance = 0.0122 µg/cm2 . The validity of the proposed method was validated statistically and quantitatively, as the detection limit value = 0.2799 µg/mL and the quantitative limit value =0.9330 µg/mL. The method proved to be of good accuracy and precision, as the recovery rate was %Rec =98.05, and the relative standard deviation %RSD did not exceed 0.42 . The formation of these particles was tested using FT-IR technology and scanning electron microscopy (SEM), and the binding of the drug to the nanoparticles was confirmed by using UV-VIS spectroscopy