Abstract: 【Objective】Bioinformatics analysis was performed on the NS1 protein of Tembusu virus（TMUV），the NS1 protein was expressed in prokaryotes，and mouse-derived NS1 polyclonal antibody was prepared，providing theoretical basis for TMUV detection and research on the function of NS1 protein.【Method】ProtParam，ProtScale，TMHMM-2.0，SignalP-5.0，NetPhos 3.1，NetNGlyc-1.0，YinOYang-1.2，SOPMA and AlphaFold2 bioinformatics softwares were used to analyze the amino acid composition，theoretical isoelectric point，hydrophilicity/hydrophobicity，transmembrane domain，signal peptide，phosphorylation site，glycosylation site，secondary structure and tertiary structure of NS1 protein. The NS1 gene was amplified，the recombinant plasmid was constructed，NS1 recombinant protein was expressed in Escherichia coli BL21（DE3）receptor cells. NS1 protein was purified and concentrated by Ni²⁺-NTA affinity chromatography after urea treatment，and then identified by SDS-PAGE and Western blotting. Mouse-derived NS1 polyclonal antibody was prepared，and an indirect ELISA method was established to detect the titer and specificity of mouse-derived NS1 polyclonal antibody.【Result】The bioinformatics analysis showed that the NS1 protein was a stable hydrophilic protein，comprised by 320 amino acid residues，with a molecular weight of approximately 36.1 kD and a theoretical isoelectric point of 8.24. The NS1 protein lacked transmembrane domain and signal peptide，contained 42 phosphorylation sites （22 serine，17 threonine and 3 tyrosine residues）as well as 13 glycosylation sites（3 N-glycosylation sites and 10 O-glycosylation sites）. In the secondary structure of the NS1 protein，α-helix constituted 21.88%，extended strands constituted 25.00%，β-turns constituted 11.87%，and random coils constituted 41.25%，and the tertiary structure consisted of 2 domains. The prokaryotic expression vector pET-28a-NS1 was successfully constructed，and NS1 protein was efficiently purified. The prepared mouse-derived anti-NS1 polyclonal antibody exhibited a high titer of 1∶3276800 as determined by indirect ELISA，it could also be used for the specific detection of TMUV and indirect immunofluorescence analysis. 【Conclusion】The NS1 protein lacks signal peptides and transmembrane domains，and is a hydrophilic protein suitable for expression in prokaryotic system. The pET-28a-NS1 is successfully constructed using a prokaryotic expression system. Following urea denaturation，the NS1 protein is purified by Ni²⁺-NTA affinity chromatography，yielding a protein with high purity and biological activity. A mouse-derived anti-NS1 polyclonal antibody with high titer and strong specificity is successfully prepared，enabling its use for the specific detection of TMUV.