Bluetongue (BT) is a non-contact infectious disease of domestic or wild ruminants caused by the Bluetongue virus (BTV). It is transmitted by Culicoides biting midges. BTV mainly infect sheep, and animals infected with BTV usually show clinical symptoms such as fever, mucosal edema, and ulcers. BTV virus is an icosahedral symmetric RNA virus with 27 different serotypes. BTV consists of 7 structural proteins (VP1-VP7) and 4 non-structural proteins (NS1, NS2, NS3/NS3a, NS4, NS5). The VP3 encoded by the L3 gene is relatively high conserved structural protein, and constitute the inner symmetric icosahedral core shell of virus particle. In this study, VP3 recombinant protein of bluetongue I virus was successfully expressed and purified by prokaryotic expression system. Moreover, to increase the amount of soluble protein, we used chaperone protein ptf16 and two fusion proteins NusA and TRX. The results show that chaperone protein can increase the solubility of VP3. Then the expression conditions were optimized and VP3 was purified by nickel affinity chromatography. The VP3 immunize Balb/c mice and the results show that the serum titer is 1:1.28×105. In the Dot-ELISA assay, we found recombinant protein VP3 react with the serum from immunized mice by inactivate BTV-1. The results of IFA showed that the antibody produced by immunized mice with recombinant protein VP3 could react with the VP3 protein expressed in 293T cell. In conclusion, we expressed the recombinant protein VP3 with the same conformation as eukaryotic expression system and had same immunogenicity with inactivated virus, which laid a foundation for further study on the structure and function of BTV.