In this study, tylosin (TYL) was derivatized with 4-aminophenylacetic acid to synthesise a new hapten and the hapten was used to produce the monoclonal antibody. The obtained antibody simultaneously recognised TYL, tilmicosin, acetylisovaleryltylosin and the metabolite of TYL (desmycosin) with crossreactivities of 100%, 62%, 97% and 93%, respectively. After evaluation of two coating antigens, a heterologous competitive indirect enzyme linked immunosorbent assay was developed to determine the four analytes in milk simultaneously. The limits of detection for the four analytes were in the range of 1.5Á3.1 ng mL (1. The recoveries from fortified milk were in the range of 76.3Á97.4% with coefficients of variation of 5.3Á15.7%.