Objective：To develop an amplified luminescent proximity homogeneous assay linked immunosorbent assay（AlphaLISA） method for simultaneous determination of clenbuterol（CLB） and ractopamine（RAC） in animal tissues. Methods：The samples were subjected to enzymatic extraction with β-glucuronidase/sulfatase and ammonium acetate，degreased with n-hexane，and filtrated with a filter membrane. Then the filtrate underwent reactions with biotinylated antigen，antibody，donor beads，and acceptor beads；subsequently AlphaLISA was used for determination. Results：CLB and RAC showed a good linear relationship in the range of 0.1 to 50 ng/mL（R2>0.98），with a limit of detection of 0.02 ng/mL. The recovery rates of CLB + RAC（1∶1），CLB，and RAC at spiking levels of 5，10，and 20 μg/kg were 88.8%-102.8%，86.6%-98.3%，and 84.2%-103.2%，respectively；the relative standard deviations were all less than 12%. Conclusion：The method developed is simple，rapid，accurate，and reliable，thus making it applicable for the determination and screening of CLB and RAC in animal tissues.