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.