Specific single-chain variable fragment (scFv) against Cronobacter was generated using genetic engineering. Genes coding for variable regions of the heavy and light chains (VH and VL) were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA from hybridoma cells secreting Cronobacter monoclonal antibody. VH and VL were fused together by Splice Overlap Extension (SOE)-PCR with flexible polypeptide linker (Gly4Ser)3 to construct the scFv genetic fragment. The scFv-pET-26b recombinant plasmid was constructed by double-digestion with restriction endonucleases XhoI and EcoRI, followed by cloning into plasmid vector pET-26b. Positive clones were selected for extraction of scFv-pET-26b, which was transformed into Escherichia coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was purified by His affinity chromatography and identified by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). Genetically engineered strains expressing Cronobacter scFv were successfully constructed. SDS-PAGE and ELISA results showed that the molecular weight of the expressed scFv was approximately 30 kDa and that the obtained scFv could specifically bind to Cronobacter and is a potential candidate antibody for the detection of Cronobacter.