The anti-ractopamine monoclonal antibody and anti-clenbuterol monoclonal antibody were both coupled with CNBr activated Sepharose 4B to make the immunoaffinity column.The column performance was tested and the use conditions were optimized by HPLC-MS/MS.The results showed that the column capacity to ractopamine,salbutamol and clenbuterol were 49,27,24 ng/mL glue,respectively,the storage time was five months,and the best eluent was methanol.The average recoveries of three lean meat essences ranged from 78.9% to 96.5% with relative standard deviations(RSD) lower than 1.5%.The limits of detection(LOD) ranged from 0.1 ng/g to 1 ng/g.The method could be applied in the simultaneous determination of the lean meat essence contents in feed samples.