Abstract: The β₂-agonist, salbutamol (SAL) was linked to bovine serum albumin(BSA) using the mixed anhydride method and the anti-SAL serum was acquired in rabbits against the conjugate. The optional dilution of the anti-SAL serum was 1∶6 400 by the indirect enzyme linked immunosorbent assay (ELISA) and the anti-SAL serum has the cross-reactivity of 110% with Clenbuterol (CL). Then an indirect competitive ELISA, which can detect SAL and CL simultaneously, was developed. The limit of detection for SAL was 0.48 ng/mL with the range of detection 0.48 ng/mL~8.0 μg/mL and the limit of detection for CL was 0.43 ng/mL with the range of detection 0.43 ng/mL~7.3 μg/mL. The recovery rates ranged from 72.2% to 84.8% for detecting SAL fortified level of 1~50 ng/g (ng/mL) in swine liver and swine urine, the coefficients of variation was 4.2%~28.3%; the recovery rates ranged from 84.3% to 103.4% for detecting CL fortified level of 1~50 ng/g (ng/mL) in swine liver and swine urine, the coefficients of variation was 0.3%~35.5%.