Vibrio parahaemolyticus, a halophilic gramnegative bacterium, is a food-borne pathogen that largely inhabits marine and estuarine environments, and poses a serious threat to human and animal health all over the world. The hollow "needle' channel, a specific assemble of T3SS which exists in most of gram-negative bacteria, plays a key role in the transition of virulence effectors to host cells. In this study, needle protein VP1694 was successfully expressed and purified, and the fusion protein Trx-VP1694 was used to immunize Balb/c mice. Subsequently, a phage single-chain fragment variable antibody (scFv) library was constructed, and a specific scFv against VP1694 named scFv-FA7 was screened by phage display panning. To further identify the characters of scFv, the soluble expression vector pACYC-scFv-skp was constructed and the soluble scFv was purified by Ni2? affinity chromatography. ELISA analysis showed that the scFvFA7 was specific to VP1694 antigen, and its affinity constant was 1.07 9 108 L/mol. These results offer a molecular basis to prevent and cure diseases by scFv, and also provide a new strategy for further research on virulence mechanism of T3SS in V. parahaemolyticus by scFv.