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A novel bi-hapten immunogen was prepared by coupling amoxicillin and chlortetracycline to bovine serum albumin in turn. Then the immunogen was used to immunise New Zealand rabbits to produce the polyclonal antibody. The obtained antibody simultaneously cross-reacted with six penicillins (amoxicillin, penicillin G, ampicillin, penicillin V, carbencillin and sulbenicillin) and four tetracyclines (tetracycline, oxytetracycline, doxycycline and chlortetracycline) with crossreactivities in a range of 24–100%. Then a broad specificity enzyme linked immunosorbent assay was optimised for the simultaneous detection of the 10 drugs in milk. The limits of detection for these analytes were in a range of 0.4–3.7 ng/mL depending on the compound. The intra- and inter-assay recoveries of the 10 analytes from standards fortified blank milk ranged from 80.8 to 99.4% with coefficients of variation lower than 13%. This study provided a new strategy for the development of multi-analyte immunoassay through immunogen design. [ABSTRACT FROM AUTHOR]