Abstract: The anti-clenbuterol monoclonal antibody ( CLMcAb) was purified by the aqueous two-phase systems ( ATPS) , and its affinity with the second enzyme-labeled antibody was taken as evaluation indexes to optimize the immobilized process for CLMcAb. Furthermore, the stability of immobilized CLMcAb could be characterized by resist erode test and atomic force microscope ( AFM) . The results suggested that the optimum CLMcAb activity could be obtained with the fixed time of 90min, the 2000 fold dilution of CLMcAb and the ratio of solid to liquid of 0.65mL per carrier at 4℃.The immobilization of CLMcAb in PEG phase resulted in the highest activity and stability by reducing the surface roughness ( Ra) significantly and forming network micelle on the carrier surface.