A new synthesis way of haptens for tetracyclines (tetracycline, chlortetracycline and oxytetracycline) based on the formation of their carboxamido and diazo derivatives that maintain the tetracyclic nucleus and possess an aromatic or aliphatic spacer arm--in order to create a degree of heterology in the chemical structure--is described. The haptens were used to successfully raise polyclonal antibodies and a reliable and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) for tetracycline was set up. After the optimization of physical and chemical parameters (pH, salt, detergent concentration, and time), a detection limit of 0.4 ng mL(-1) for tetracycline was reached. The optimized ELISA showed cross-reactivity to rolitetracycline (91%), oxytetracycline (30%), methacycline (14%), and chlortetracycline (10%). The analytical potential of the immunoassay was demonstrated by the excellent recoveries obtained (mean value ranging between 79% and 108%) when determining tetracycline residues in honey samples with a simple preparation procedure. The obtained results confirmed the suitability of the assay as screening method for either food monitoring or laboratory quantification of tetracycline residues.