Transient　isotachophoresis　with　field-amplified　sample　injection　(FASI),　using　β-CD　as　the　chiral　selector　and　tetrabutylammonium　hydroxide　(TBAOH)　as　the　additive,　was　applied　for　on-line　preconcentration　and　enantioseparation　of　three　β-agonists,　namely,　cimaterol,　clenbuterol　and　terbutaline.　The　experimental　conditions　for　both　simultaneous　enantioseparation　and　on-line　preconcentration　methods　have　been　investigated　in　detail.　Under　the　optimum　conditions,　the　detection　limits　(defined　as　S/N　=　3)　of　this　method　were　found　to　be　1　ng　mL-1　for　all　three　pairs　of　β-agonists　enantiomers.　Compared　with　conventional　electrokinetic　injection,　the　enhancement　factors　were　greatly　improved　to　be　250-fold.　Finally,　the　proposed　method　has　been　applied　for　the　analysis　of　human　urine　samples.　©　2011　The　Royal　Society　of　Chemistry.