Abstract: Classical Swine Fever Virus （CSFV）E0 gene was amplified from the plasmid pMD18-T-E0 by PCR and cloned into the adenoviral shuttle plasmid pAdTrack-CMV. A two-step transformation protocol was employed for the construction of recombinant adenoviral plasmid pAdEasy-E0. The resultant recombinant DNA was transfected into 293 cells to yield packaged adenovirus. E0 gene was confirmed to be integrated into the genome of recombinant adenovirus by PCR, and the expression of E0 was detected in the 293 cells infected with recombinant adenovirus by Western blot. ELISA results showed that systematical immune responses to CSFV virus could be induced effectively in mice after twice immunizations through intraperitoneal route. The replication-defective recombinant adenovirus expressing (CSFV) E0 was constructed and could elicit favorable immune responses in mice.