Abstract: A highly active antibody was produced by making manmade antigen of Clenbuterol.We established a direct competition method to detect Clenbuterol that used HRP as markers and luminol as chemiluminescent substrates, with which many parameters were optimized, including coating buffer, coating condition, blocking condition, incubating time, and enzyme consumption.Based on this method we developed a chemiluminescent detecting kit, whose linear range was 0.1-8.1ng·mL-1, the linear correlation coefficient was r=0.996, the IC50 was 0.607ng mL-1, the detection limit was under 0.1ng·mL-1, the within-run assay coefficient of variation was 6.76%-8.31%, the between-run assay coefficient of variation was 7.4%, the coefficient of recovery was between (90±15) %, and there was no cross reaction with otherβ-agonists.