In　order　to　establish　the　immunoassay　method　of　Chloramphenicol　(CAP),　CAP　was　coupled　with　carrier　protein　bovine　serum　albumin　(BSA)　to　prepare　complete　antigen　CAP-BSA　by　diazotization　reaction.　And　optimize　the　coupling　condition　by　quadratic　regression　revolution　design.　The　complete　antigen　was　identified　by　ultraviolet　spectrum,　infrared　spectrum　analysis.　Results　indicated　that　synthesis　of　CAP　antigen　was　successful.　The　coupling　ratio　was　18　and　concentration　of　protein　was　2.53　mg/mL.　Polyclonal　antibodies　against　chloramphenicol　are　produced　from　mouse,　titrations　of　serum　was　determined　by　methods　of　protein　SDS-PAGE　and　indirect　ELISA.　After　the　forth　immunization,　the　titer　of　M2　reached　218　700　by　methods　of　indirect　ELISA.　This　experiment　will　provide　a　more　rapid　and　stable　method　for　the　quantitative　analysis　of　chloramphenicol　in　future.