Immunoassay for determining aflatoxin B 1 involves in using the toxic mycotoxin in a conjugated form. To replacing the toxic conjugate in an immunoassay, four mimotope peptides of aflatoxin B 1 were acquired by a panningelution selection from a phage library, and one mimotope peptide was fused with the major coat protein gVIIIp by the pC89 phagemid display system. It led to a high copy number expression of the mimotope peptide in a recombinant phage. The recombinant phage was identified by an anti-aflatoxin B1 monoclonal antibody, and confirmed by DNA sequencing. An immunoassay was set up with the recombinant phage. The new method was compared to a conventional immunoassay. The calibration curves and the results of accuracy and precision were almost identical in both methods, which demonstrated the possibility of using the recombinant phage replacing the aflatoxin B 1 -protein conjugate to set up immunoassay.