A liquid chromatography–linear ion-trap spectrometry (LC-MS3) method using β-receptor molecular-imprinted polymer (MIP) solid-phase extraction (SPE) as clean-up was developed to determine simultaneously and confirmatively residues of 25 β2-agonists and 21 β-blockers in urine samples. Urine samples were subjected to enzymatic hydrolysis by β-glucoronidase/arylsulphatase, and then extracted with perchloric acid. Sample clean-up was performed using β-receptor MIP SPE. A Supelco Ascentis® express Rp-Amide column was used to separate the analytes, and MS3 detection used an electrospray ionisation source in positive-ion mode. Recovery studies were carried out using blank urine samples fortified with the 46 analytes at the levels of 0.5, 1.0 and 2.0 μg l–1. Recoveries were obtained ranging from 60.1% to 109.9% with relative standard deviations (RSDs, n = 7) from 0.5% to 19.4%. The limits of detection (LODs) and limits of quantitation (LOQs) of the 46 analytes in urine were 0.02–0.18 and 0.05–0.60 μg l–1, respectively. As a result of the selective clean-up by MIP SPE and MS3 detection of the target drugs, the sensitivity and accuracy of the present method was high enough for monitoring β2-agonist and β-blocker residues in urine samples. Satisfactory results were obtained in the process of the determination of positive urine samples.