An amplified luminescent proximity homogeneous assay(AlphaLISA) was developed for the determination of four kinds of β agonists(clenbuterol,salbutamol,brombuterol,terbutaline) in pork.The analytes were stepwise extracted with ammonium acetate and β-glucuronidase/sulfatase,the extract was columned and concentrated to dryness,redisolved in buffer.Samples were reacted with botinylated antigen,antibody,donor beads and acceptor beads,and detected by AlphaLISA with matrix standard curve.As a result,there are good linear relationships in the concentration range of 0.1-500 ng/mL，with correlation coefficients larger than 0.98.The cross-reactivities for clenbuterol,brombuterol,terbutaline were 143.3%,179.2%and 95.6%,respectively,and the limits of detection(LOD) were 0.03-0.08 ng/mL.The average recoveries of four compounds at spiked levels of 0.5,10,20 μg/kg were in the range of 82.5%－115.9%with relative standard deviations(RSD) not less than 12.0%.The method is simple,rapid and accurate,and is suitable for the detection and screening of β-agonists in pork.