A novel method of polymer monolith microextraction (PMME) using poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolith combined with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF MS) was developed for the rapid and sensitive determination of β-agonists in pork samples. The conditions of PMME were optimized for the improvement of extraction efficiency and reduction of the matrix interferences from pork. Under the optimal condition, the eluate solution allowed direct analysis by mass spectrometry. In the positive ion mode and in the multiple reaction monitoring (MRM) mode, the limits of detection (LODs) for β-agonists were found to be 0.08 ng/g (clenbuterol, CLB), 0.18 ng/g (salbutamol, SBTM) and 0.26 ng/g (terbutaline, TBTL) in pork, respectively, with good inter- and intra-day precisions (2–10% for CLB, 11–23% for SBTM and 4–16% for TBTL). The proposed PMME/ESI-QTOF MS method was successfully applied to the determination of β-agonist residues in thirteen real samples, and the positive samples were confirmed according to the identification points (IPs) system defined by Commission Decision 2002/657/EC. To investigate the matrix effect, the proposed method was compared with PMME-HPLC/ESI-QTOF MS and the slight decrease in sensitivity of PMME/ESI-QTOF MS was ascribed to the inter-analyte ion suppression. Copyright © 2007 John Wiley & Sons, Ltd.