A　method　using　hollow　fibre-protected　liquid-phase　microextraction　(HF-LPME)　with　in　situ　derivatization　followed　by　gas　chromatography/mass　spectrometry　(GC/MS)　was　established　for　the　analysis　of　β-agonists　and　β-blockers　in　urine.　Because　it　can　simultaneously　extract　and　derivatize　compounds　of　interest　by　methylbenzol　and　N-methyl-N-(trimethylsilyl)　trifluoroacetamide　(MSTFA)　in　HF-LPME,　the　approach　overcomes　the　drawbacks　of　considerable　time-consuming　and　tedious　operation,　meanwhile　improves　enrichment　multiple.　The　optimized　conditions　were　extraction　for　20　min　at　35　°C　with　5.0　μL　of　mixed　extraction　solvent　(methylbenzol/MSTFA　=　1:1,　v/v)　with　stirring　speed　of　925　rpm　in　5.0　mL　sample　under　pH　12.0　and　14%　(w/v)　NaCl.　The　method　provided　very　wide　linear　ranges　(0.25-400　ng　mL-1)　and　low　detection　limits　in　the　range　of　0.08-0.10　ng　mL-1　for　clenbuterol,　metoprolol　and　propranolol　while　enrichment　factors　reached　up　to　256.　The　analytes　could　be　determined　in　spiked　urine　by　the　method　with　high　extraction　efficacy　(93.79-109.04%　recoveries)　and　precision　(<9.70%　RSD).　It　has　a　satisfactory　result　for　metoprolol　in　practical　human　urine　samples　for　a　single-dose　administration　of　50　mg　after　36　h.　The　proposed　method　only　needs　few　microliters　of　organic　solvent　and　derivatizing　agent;　the　operation　is　simple,　convenient　and　rapid　for　the　trace　analysis　of　β-agonists　and　β-blockers　in　biological　fluids;　it　can　be　readily　generalized　for　high　sample　throughput.　So,　it　is　hopeful　that　the　study　will　facilitate　the　monitoring　of　β-agonists　and　β-blockers　in　the　competition　sports.　©　2009　Elsevier　B.V.　All　rights　reserved.