Abstract: Objective: : A human single-chain antibody library was constructed from lymph nodes of esophageal cancer patients using phage display techniques. The aim of this study was to screen and identify the human single-chain variable fragment(scFv) associated with esophageal cancer and to detect the activity of the antibody. Methods : The insert ratio of the scFv antibody library was identified by PCR. Positive insert clones wereidentified by SfiI/NotI double digestion followed by 1.5% agarose gel electrophoresis. Normal humanesophageal epithelial cells(NHEEC) and esophageal cancer cell line Eca109 were used to screen the phageantibody library. Strongly positive recombinant phage clones were used to infect E. coli HB2151 and induceexpression of soluble scFv. Soluble scFv from periplasm was purified by affinity chromatography. The relativemolecular weight of the scFv was detected by SDS-PAGE. Western blot, immunohistochemistry, immunocytochemistry and ELISA were used to characterize the activity of the antibody. Results : Enzymatic digestionsshowed that the positive insert ratio was 91.7%(22/24). Agarose gel electrophoresis showed that target fragments were released as products of SfiI/NotI double digestion of positive insert clones. Recombinant scFvphage had a titer of 3.3×1011 cfu/mL. The fourth phage harvest yielded 141 times as much as that of the firstone. The results of SDS-PAGE and Western blot showed that the MW of the soluble scFv was about 30kD.Tissues from esophageal cancer showed strong immunohistochemical staining and the esophageal cancercell line Eca109 showed strong immunocytochemical staining with the recombinant scFv. ELISA results revealed that soluble scFv was highly active and could bind Eca109 cells, but not BGC-823 or NHEE cells. Conclusion : A human phage-display antibody library was successfully produced using esophageal cancer cellsfound in lymph nodes. The scFv antibody fragment specifically binds esophageal cancer cells.