Abstract: In order to improve the stable isotope utilization rate, a simple and inexpensive synthesis method was developed. The synthesis of levofloxacin is based on piperazine as the starting material, and the reaction with carbobenzoxy chloride (CBZ-Cl) to get CBZ-piperazine-CD₃, and then the reaction with iodomethane-D₃ to get CBZ-piperazine-CD₃, after the removal of CBZ protection to get methylpiperazine-D₃, and finally the reaction with levofloxacin carboxylic acid to get levofloxin-D₃. The yield of levofloxacin-D₃ was 37.0% based on the amount of iodomethane-D₃. The synthesized Levofloxacin-D₃ product was characterized by nuclear magnetic resonance spectroscopy, high performance liquid chromatography and mass spectrometry. The chemical purity was 98.5% and the isotope abundance was 99.5atom% D. It can be used as the internal standard for the quantitative determination of levofloxacin residue in animal food.