Abstract When pigs are infected with classical swine fever virus (CSFV), the antibody primarily targets the structural gly-coprotein E rns of the virus. Previous investigations have demonstrated that E rns has low or no virus neutralizing capacity. In this study, candidate subunit marker vaccine, chaperonin 10 (Cpn10)-E rns, which possess the property of generating neutralized antibodies against lethal challenge of virulent CSFV was developed. The gene of E rns was isolated from Hog cholera lapinized virus (HCLV) -infected spleen cells of rabbits via RT-PCR method and fused to the downstream region of the cpn10 gene; the products of recombinant fusion protein (cpn10-E rns) induced expression in Escherichia coli, and the products were purified by affinity chromatography. During the course of vaccination, the candidate vaccines cpn10-E rns were used for the immunization of guinea pigs, and they induced a strong antibody response against cpn10-E rns. The antibodies can be immobilized by coating inactivated CSFV particles, indicating that these antibodies can recognize CSFV. Neutralization assay was carried out on rabbits according to National Regulations on Veterinary Drug. The results clearly indicate that the typical fever of rabbits induced by the live attenuated HCLV could be inhibited by preincubation with the antisera (dilution 1:4) induced by cpn10-E rns, but not inhibited by preincubation with the antisera induced only by E rns. Analogous results were observed for the group of the rabbits immunized with cpn10-E rns, which were protected against the typical fever induced by the challenge with HCLV. The findings of this study formed the basis of a new means for developing subunit marker vaccine against CSFV.