Objective:To construct a plasmid pET28-NS1 for expression of nonstructural protein(NS1) of murine influenza virus FM1 in E.coli and utilize the NS1 protein in serological detection for differentiating infected from vaccinated mice. Methods:The NS1 gene of murine influenza virus A/FortMonmouth/1/47(FM1) was amplified by RT-PCR and cloned into pET28a to construct a recombinant expression plasmid, named pET28-NS1. The constructed vector was identified by PCR, enzyme digestion and nucleotide sequences analysis after being transformed into E.coli BL21. Induced by IPTG, the recombinant protein was analysed by Western blot and purified with Ni+ affinity chromatography. Based on the purified recombinant protein, an indirect ELISA assay for detection of anti-NS1 protein antibody(NS1-ELISA) was developed. The sera of virus-infected mice and vaccinated mice were detected at 7,15 and 30 day, respectively, and data were analyzed by SPSS13.0. In the end, The new assay was methodologically evaluated by sensitivity, specificity, positive and negative predictive value. Results:The recombinant plasmid was constructed correctly. SDS-PAGE and western blot analysis showed that the recombinant protein was highly expressed by inclusion body and its molecular weight was about 26 ku as expected. NS1-ELISA indicated that the purified protein reacted with the sera of mice experimentally infected with FM1, but not with those of the mice immunized with the inactivated viruses. Conclusion:The result demonstrated that the NS1-ELISA assay could differentiate infected from vaccinated mice on the basis of anti-NS1 antibody.