To prepare Citrinin(CIT)-protein antigen, CIT was conjugated with bovine serum albumin (BSA) by 1,4-butanediol diglycidyl ether. HPLC, UV and IR absorption suggested that CIT was correlated with the carrier protein, and the molar ratio of CIT to BSA was 8.16. The polyclonal antibodies (PcAb) against CIT were produced in serum of immunized BALB/C mice with CIT-BSA, and the titer of antibody reached to 1.1×10⁵ by indirect enzyme-linked immunoassay. The indirect competitive ELISA showed that the detection limit of CIT was 10 μg/L, with a good linearity ranging 10～250 μg/L, and IC₅₀ was 100 μg/L. Immunogenicity of antigens prepared by different methods was analyzed. The result showed that the epitope was the carboxyl group at C₇. This work would be helpful for establishing the technology and developing the kit to determine CIT-contaminated samples by ELISA.