Abstract: In order to develop FCoV diagnostic kits and vaccines based on S protein, the experiment used bioinformatic analysis to analyze the expression of FCoV S protein, to determine the expressible region of protein containing antigenic epitopes, to express recombinant protein in prokaryote in Escherichia coli, to screen the expression conditions of recombinant protein, and to identify the immunogenicity of recombinant protein. The results showed that the α-helix, extension chain, β-angle and irregular coil accounted for 25.82%, 28.14%, 4.23% and 41.8% respectively in the secondary structure of type Ⅰ FCoV S protein. There were 4 obvious hydrophilic regions and 1 obvious hydrophobic region. The protein had a transmembrane helix structure and signal peptide, and there were 3 N-glycosylation sites with a high probability and relatively exposed 46-amino-acid fragments on the surface of S protein. The S protein 1 127-1 400 aa fragment was used as the fragment of in vitro expression target and named S_1127-1400aa. The optimal induction temperature of recombinant protein was 37 ℃; the optimal induction concentration of IPTG was 0.1 mmol/L, and the optimal induction time was 5 h. Recombinant proteins were inclusion body expression; successful purification of S_1127-1400aa protein was carried out, which had good immunogenicity with FCoV-positive serum. The results suggested that the FCoV S protein fragment was successfully expressed and purified in this experiment, and the protein had good immunogenicity.