This study aimed to develop a monoclonal antibody based icELISA method for Ractopamine (Rac) residue. For this purpose, mixed anhydride method was employed to synthesize the immunogen of Rac-BSA and 1, 4-butanediol diglycidyl ether was used to prepare the coating antigen of Rac-OVA, thus pursue the heterologous sensitivity. Through cell fusion technology, four Hybridoma named R1-B5, R2-B3, R2-C6, and R4-C8 were screened out, and the Kas of all mAbs were between 2.7 and 4.8×109 L/mol. Based on the R1-B5 mAb, a heterologous icELISA standard curve was developed. The working range was from 0.013 to 33.7 ng/mL, with LOD and IC50 value of 0.007 ng/mL and 0.67 ng/mL, respectively. Therefore, this icELISA can be used for detecting Rac residue in animal products.