Abstract: A method has been developed for the simultaneous determination of 9 β-lactamantibiotics residues in milk by high-performance liquid chromatography- ultrasonic assisted extraction (HPLC-UAE).The milk samples were extracted with acetonitrile (phosphate buffer solution).The target analytes were extracted by 20Hz Ultrasonic-assisted extraction (UAE) for 5minutes and centrifuged,then cleaned-up with solid-phase extraction column.The chromatography was performed by Agilent TC C18 (150mm×4.6mm id,5μm) column.After it was washed by phosphate buffer-methanol,flow rate 1.0mL/min,220nm detection,9 β-lactam antibiotics were separated within 22min.The method can be used simultaneous determination of aforementioned β-lactam antibiotics in milk.