In this study,we constructed a phage display antibody library specific for gentamicin(GEN) to get the anti-getamicin single chain antibody(scFv).We used the anti-gentamicin hybridoma(2A3) as a source of gene to produce full-length scFv fragment.The full-length scFv fragment was ligated into the phagemid vector pCANTAB5E.Then the phagemid was transformed to E.coli TGl cells by electroporation.The size of antibody libraries was 6.5×106.The primary phage display library was riched by stabilized antigen immunoaffinity panning with GEN-OVA conjugates.9 clones congtained insert and showed GEN-positive in phage-ELISA,which lay the foundation for further immunological analysis.