Introduction: Interest in methods to improve the efficiency of animal production has focused on betaadregenic agonists such as cimaterol in recent years. Cimaterol has been recognised as a repartitioning agent that improves carcass composition by increasing muscle deposition and reducing fat. It also has the ability to accelerate livestock growth. In human however, they are generally used as anti-asthmatic agent. Therefore, excess cimaterol residues in livestock products may pose a genuine risk for human health. This has led to the prohibition of cimaterol usage in every stage of food production. To know whether food matrices contain cimaterol residue, one must need to test it. Objective: To develop a sensitive and specific Enzyme-linked Immunosorbent Assay (ELISA) for the detection of cimaterol in various matrices. Methods: The antiserum was raised in rabbits, employing cimaterol-diazo-BSA (CIM-BSA) as antigen. The ELISA test is a solid phase direct competitive assay using horseradish peroxidase conjugate as the competing entity. The total incubation time of the assay is 45 minutes. Results & Discussion: The concentration of cimaterol to decrease tracer binding by 50% (IC50 value) in a direct competitive ELISA method was found to be 2.9ng/mL. The antibody is specific with low crossreactivity towards other beta-agonists tested such as propanolol (11.7%), clenbuterol (11.4%), mapenterol (7.3%), metoprolol (5.3%), and atenolol (4.6%). The antibody has cross-reactivity of < 1% to other drugs such as terbutaline, ractopamine, timolol and isoxsuprine. The limits of detection (LOD) of the ELISAfor swine urine, chicken muscle, shrimp tissue, milk and animal feed were determined as 0.33 ng/mL, 0.30 ng/mL, 0.30 ng/mL,1.18ng/mL, and 0.28 ng/mL respectively. Conclusion: A sensitive and specific ELISA was developed to detect the beta 2-adregenic agonist cimaterol in various matrices such as urine (swine), muscle (chicken), tissue (shrimp), milk and animal feed.