Abstract: The electrochemical behaviors of oxytetracycline on Cu₂O microspheres modified glassy carbon electrode were studied, and it was found that the oxidation of oxytetracycline was an adsorption-controlled, one-proton, one-electron, and irreversible process. In pH 4.8 HAc-NaAc buffer solution, an electrochemical method with high sensitivity was developed for the determination of oxytetracycline by using square wave voltammetry after an open-circuit accumulation at 0.4 V. The linear range was 0.5~210 μmol/L and the limit of detection was 0.15 μmol/L (R_SN=3). Finally, this method was applied for the determination of oxytetracycline in animal-derived food, and the recoveries were between 97.4%~107%.