Abstract: Objective To amplify and express the gene encoding murinc single-chain fragment variable (scFv) antibody against human cervical cancer and predict the secondary structure and three-dimensional structure of the antibody. Methods The gene fragments coding for the variable region of the heavy and light chains of scFv antibody against human cervical cancer were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, and spliced together using a flexible linker to the antibody. The scFv genes were then cloned into the expression vector pCANTAB 5E and expressed in E.coli HB2151 and TG1, respectively. The scFv antibody obtained was assayed using SDS-PAGE, Western blotting, and immuno-histochemical analysis, with its secondary structure and three-dimensional structure predicted with Swiss-model and 3dpssm. The physicochemical properties of the antibody were analyzed with Antheprot software. Results The expressed scFv antibody was soluble and phage-displayed. The specific binding capacity of the soluble and phage-displayed scFv antibody to the surface-associated antigen of human cervical cancer cell line were further confirmed. The scFv antibody had a relative molecular mass of 32 000 as was consistent to theoretical prediction, with pI of 7.215 and characterized to belong to α+βprotein. The antibody contained many protein kinase C phosphorylation sites and casein kinase Ⅱphosphorylation sites in both the VH and VL domains. Computer graphic modeling indicated that the linker was isolated from the VH and VL domains, which formed a hydrophobic pocket in the molecule to facilitate antigen binding. Conclusions The soluble and phage displayed scFv antibody expressed in E.coli against human cervical cancer shows high and specific affinity to the cervical cancer cell line surface-associated antigen. The construction and analysis of the molecular model of the antibody may facilitate further studies in engineering of the antibody and exploration of the mechanism of the antigen-antibody interaction.