A　method　was　developed　to　perform　the　screening,　quantification　and　confirmation　of　the　five　β-blockers,　propranolol,　carteolol,　bisoprolol,　esmolol,　and　sotalol,　in　human　urine　using　gas　chromatography-mass　spectrometry　(GC-MS).　In　sample　preparation,　conjugated　and　unconjugated　β-blockers　in　urine　were　extracted　separately,　and　the　extracts　were　combined.　The　extracts　were　derivatized　with　N-methyl-N-trimethylsilyl-trifluoroacetamide　(MST-FA)　and　N-methyl-bis(trifluoroacetamide)　(MBTFA).　The　optimal　conditions　of　GC-MS　were　established,　and　the　progresses　of　screening　by　selected　ion　monitoring　(SIM)　mode　and　confirmation　by　full　scan　(SCAN)　mode　were　completed.　At　last,　the　quantification　curves　of　the　five　β-blockers　in　spiked　urine　were　established　by　SIM　mode.　The　limits　of　detection　were　0.　2　-1.0　ng/mL.　Overall　recoveries　were　70.5%　-　103.4%,　and　the　relative　standard　deviations　were　lower　than　15%.　In　addition,　the　method　was　successfully　applied　to　the　analysis　of　the　positive　urine　of　propranolol,　and　the　urinary　excretion　curve　was　also　established　accordingly.　It　is　significant　to　prohibit　the　abuse　of　β-blockers　in　doping　control.