Objective To establish a noncompetitive and homogeneous immunoassay for chloramphenicol based on the antigen-dependent reassociation of antibody variable fragment and β-galactosidase ( β-gal ) complementation ( open sandwich enzymatic complementation immunoassay，OS-ECIA) ，chloramphenicol-driven complementation fragment of β-gal were constructed. Method Coding sequences of Δα and Δω of β-gal were amplified from genomic DNA of Escherichia coli cell lines DH5α and BL21， respectively， then fused to antibody variable region gene ( V_H-CAP or V_L-CAP) against chloramphenicol by a linker according to OS-ECIA model，and expressed under the control of promoter T7 in E. coli. Inclusion proteins were renatured after purification and analyzed for β-gal complementation activity. Results V_H-CAP -Δα and V_L-CAP -Δω genes were cloned as designed. Both fusion peptides gene were over-expressed as inclusion proteins. Inclusion proteins were purified to a purity over 80% ，then refolded by dilution method，and both refolded fusion proteins showed typical chloramphenicol-dependent complementation activity. Coclusion chloramphenicol-driven complmentary system of β-gal were successfully constructed.