Abstract: Multiple bioinformatics tools were used to predict the Tembusu virus(TMUV) envelope E protein and its domain Ⅱ(ED Ⅱ) the transmembrane domains. Recombinant expression plasmids were constructed, expressed and purified the recombinant E and EDⅡ proteins in a prokaryotic system. Mouse-derived polyclonal antibodies against the E and EDⅡ proteins were prepared. An indirect ELISA method was established using a checkerboard titration to determine antibody titers, and the specificity of the antibodies was evaluated by Western blotting and indirect immunofluorescence assay(IFA). The results showed that:(1)Bioinformatics analysis revealed that the E protein consists of 501 amino acid residues, is a hydrophilic protein with two transmembrane regions and no signal peptide. A total of 50 phosphorylation sites and two N-glycosylation sites(154 and 314 aa) were predicted. In the secondary structure of the E protein, α-helix, extended strand, β-turn, and random coil account for 22.55%, 31.14%, 11.78%, and 34.53%, respectively. Tertiary structure prediction indicated that the E protein consists of three domains(EDⅠ, EDⅡ, and EDⅢ). The prokaryotic expression vectors pET-28a-E and pET-28a-ED-Ⅱ were successfully constructed, and the recombinant E and EDⅡ proteins were purified and verified. Mouse-derived polyclonal antibodies against these proteins exhibited high titers of up to 1:1638400 and 1:409600, respectively. Both Western blotting and IFA confirmed the excellent specificity of the antibodies.