Abstract: A method for multi-residue analysis of β-agonists, clenbuterol, brombuterol, tulobuterol, salbutamol in pork was established by using isotope dilution combined with gas chromatography-tandem mass spectrometry and based on solid phase extraction method. β-agonists, which are well-known for their ability to improve growth rate and reduce carcass fat when fed to farm animals, were often adulterated into animal feed illegally. However, this misuse had caused some severe accidental poisoning in humans. Therefore, the use of β-agonists in animal dreeding is banned in many countries, and the establishment of a high sensitivity, accurate of β-agonist detection method is very necessary. In this study, the homogenized samples were spiked with isotope-labeled internal standards clenbuterol-D₉, brombuterol-D₉, tulobuterol-D₉, enzymatically hydrolysed and extracted by acetic acid-buffer, adjusted pH by perchloric acid, and cleaned-up on HLB and MCX cartridge, and derivatized with N, O-bis (trimethylsilyl) trifluoro acetamide (BSTFA)+1% trimethylchlorosilane (TMCS), finally analyzed by GC-MS/MS. The experimental results showed that spiking with the 4 β-agonists at 2.5-35 μg/kg in blank samples, the recoveries were 92.2%-112.8%, and intra-day and inter-day coefficients were 1.0%-12.6% and 1.9%-13.2%, respectively. Using isotope as the internal standard for the determination of the material, it can effectively reduce the sample errors in the pretreatment, derivatization and instrumentation process. And under the certain conditions, 4 β-agonists can be effectively separated. The linear ranges of 4 β-agonists were 35-158.00 μg/L, the coefficient of correlation was not less than 0.999 7, detection limits were in the range of 0.13-0.4 μg/kg, and the quantitative limits were in the range of 0.40-1.27 μg/kg. The method is demonstrated to be suitable for the determination of clenbuterol, brombuterol, tulobuterol, salbutamol in pork.