The aim of this study is to prepare the ScFv antibody (single chain variable fragment antibody) against viral hemorrhagic septicemia virus (VHSV) and analyze the biological properties. The variable heavy (VH) and the variable light (VL)chain gene fragments were derived from mAb 1G5 hybridoma cells against VHSV. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in the pET28a vector to express ScFv protein in E.coli cells. The expressed ScFv protein, with a relative molecular mass of about 28 ku, existed in a form of soluble expression in cytoplasma. The ScFv protein could specifically identify VHSV glycoprotein (G), and neutralize viral virulence of VHSV in vitro. The ScFv protein showed good affinity for VHSV glycoprotein (G) antigen, as indicated by KD values of 1.4×10^-8 M. ScFv protein preparation has laid a foundation for further study of VHSV therapeutic antibodies as well as rapid diagnostic reagent.