Abstract: Objective To establish a biological method for determining the macrolide content in various matrices. Method Human serum, urine and tissue homogenate samples were diluted or extracted with MSU buffer, and the specimens of grain and premixed feed extracted with methanol-H3PO4 buffer, before microbial receptor competitive binding assay was carried out on these various specimens, with the elimination of interference from methanol with the M8 buffer. Results The method was sensitive, class-specific and precise, and the recommended screening concentrations for specimens of the serum, urine, tissue and grains were 200, 200, 100 and 1 200 ng/g (ng/ml), respectively, with a relative standard deviation less than 8%. Conclusion Microbial receptor competitive binding assay is accurate and rapid for efficient qualitative and quantitative assay of the total macrolide content in various matrices.