Abstract: In order to solve the shortage of internal standard reagents for stable isotope labeling of levofloxacin, the synthesis process of stable isotope labeled levofloxacin was studied. The synthesis of Peofloxacin is based on 3-chloro-4-fluoroaminobenzene as the starting material. It is obtained by cyclization reaction with diethyl ethoxymethylmalonate (EMME), followed by reaction with iodoethane-D₅ to obtain ethylated Peofloxacin-D₅. Then, it is hydrolyzed with methyl piperazine to obtain Peofloxacin-D₅. Calculated based on the molar mass of ethanol-D₆ input, the yield of pemofloxacin-D₅ is 36.0%. The synthesized levofloxacin-D₅ product was characterized and confirmed by ¹H NMR, ¹³C NMR, HPLC, and MS, with a chemical purity of 99.3% and an isotopic abundance of 99.6 atom% D. It meets the requirements for quantitative detection of levofloxacin residues in animal derived foods using isotopic internal standards.