A magnetic solid-phase extraction method (MSPE) was developed to pre-concentrate and cleanup clenbuterol (CLE) from pork muscle. Novel sulfonated polystyrene magnetic nanobeads (spMNBs) were synthesized via a one-pot emulsion copolymerization method by using divinylbenzene, styrene, and sodium styrene sulfonate in the presence of oleic acid-modified and 10-undecylenic acid-modified magnetic ferrofluid. The resulting spMNBs exhibited high adsorption efficiency for CLE and for 10 other common beta-adrenergic agonists, namely, brombuterol, ractopamine, tulobuterol, bambuterol, cimbuterol, mabuterol, clorprenaline, penbutolol, salbutamol, and cimaterol. The adsorption behavior of the spMNBs for CLE was described by the Langmuir equation with a maximum adsorption capacity of 0.41 mg/g. Under the optimized parameters, the extraction of CLE from 0.5g of pork muscle required 25mg of the spMNBs at a shortened adsorption time (0.5min). The proposed MSPE was coupled with colloidal gold nanoparticle-based immunochromatographic assay (MSPE–AuNPIA) for the quantitative detection of CLE residue in pork muscle. The limit of detection and limit of quantification for the pork muscle were 0.10 and 0.24ng/g, respectively. The intra-day and inter-day assay recoveries at three CLE spiked concentrations ranged from 92.5% to 98.1%, with relative standard deviations ranging from 3.2% to 13.0%. The results of MSPE–AuNPIA were confirmed by liquid chromatography-tandem mass spectrometry (LC–MS/MS). The CLE values obtained with MSPE–AuNPIA agreed with those obtained with LC–MS/MS.