Carboxyl group of Free Thyroxine(FT4) was selected as the modified site in this study,Firstly,the amino group was protected with di-t-butyl dicarbonate(Boc2O),Then FT4,modified by terminal-alkyne,was prepared with the amidation reaction between Boc-FT4 and Propargylamine catalyzed by HATU.Finally,the FT4H hapten,which contained a "spacer arm" of triazole ring conjugated to carboxyl site of FT4,was synthesized via trace cuprous salt catalyzed "Click Chemistry" between terminal alkyne and 5-azide valeric acid.Immunogen and coating antigen of FT4H were respectively prepared by coupling reaction between hapten and bovine serum albumin(BSA) or keyhole limpet hemocyanin(KLH) using the N-hydroxysuccinimide active ester method.UV spectrum of the artificial antigen(FT4H-BSA) differed significantly from corresponding spectrum of hapten(FT4H) and BSA,the result also showed that FT4H was conjugated to BSA successfully,the coupling ratio of hapten to BSA was 12 ∶ 1.The 6-8 weeks old Balb/c mice were immunized with FT4H(2 g/L,0.1 mg per rat).Antiserum was collected from tails of mice after immunized five times.The antiserum titer could be up to 1.28×105 through indirect ELISA determination.The result showed that the space arm containing triazole ring as the strong determinant couldn’t interfere the combination between antiserum and FT4,and provided a new way to prepare artificial antigen of the small molecule.