The misuse of β-agonists leads to a potential risk to public health and is forbidden in many countries. We developed a rapid, sensitive and reliable multi-residue detection method for zilpaterol, ractopamine, and clenbuterol in bovine tissues by liquid chromatography–tandem mass spectrometry. Residues were extracted in ethyl acetate after protein precipitation, and then analyzed by the developed method. Good linearities (R2 > 0.99) were observed, and the recoveries of zilpaterol, ractopamine, and clenbuterol were 99%, 74%, and 102%, respectively. The limits of quantitation for zilpaterol, ractopamine, and clenbuterol were 1.3, 5.0, and 1.7 ng/g, respectively. The method is also applied successfully to bovine tissues within the Korean National Residue Programme. None of the 3 β-agonists were detected from 50 domestic samples. However, zilpaterol (6.3 ng/g) was quantified in one out of the 50 imported samples. The application of this method will be helpful in quality control analysis of β-agonists residues in bovine tissues.