Abstract: In order to construct a recombinant adenovirus expressing NcSRS2 protein of bovine Neospora Caninum, NcSRS2 gene of bovine Neospora Caninum was amplified by PCR, pMD18TNcSRS2, pCR259NcSRS2 and TransposeAdNcSRS2 were constructed in this research. Coated with liposome, TransposeAdNcSRS2 was transfected into QBIHEK293 cells to package recombinant adenovirus Ad5NcSRS2. Recombinant adenovirus NcSRS2 gene was detected by PCR. The expression of NcSRS2 gene in QBIHEK293 cells was detected by IFAT and Western blotting. After the virus titer was determined, the virus fluid was collected to inoculate BALB/c mice and IgG antibody levels in the sera were measured by indirect ELISA. The size of NcSRS2 gene was 1 227 bp. The nucleotide sequence of the gene shared 99% homology with that in GenBank (AF061249). Recombinant adenovirus Ad5NcSRS2 was successfully packaged in 293 cells. The protein expressed by Ad5NcSRS2 was 43 kD and had good reactogenicity. The titer of recombinant adenovirus Ad5NcSRS2 was 109TCID50·mL-1. Three weeks post the second immunization, IgG antibody titers in the sera of BALB/c mice were up to 1:2 048, measured by indirect ELISA. A recombinant adenovirus Ad5NcSRS2 which have good immunogenicity was successfully constructed. This settles a solid foundation for the development of recombinant adenovirus vaccine against Neospora Caninum based on NcSRS2 gene.