Canine adenovirus type 2 (CAV-2) has been proposed as a vector for recombinant vaccine. Alternatively, it may be an attractive tool for gene transfer due to lack of pre-existing immunity in humans. In this study, a transfer vector based on CAV-2, in which the 1381bp fragment of the E3 region was deleted, and a linker containing the Not I, Cla I, Fse I restriction enzyme sites were cloned into the deleted region. The recombinant CAV-2 genome was released from the plasmids enzyme digestion and transfected into MDCK cells by lipofectamine to obtain the recombinant virus. No significant difference in morphology, hemagglutination and replication between the recombinant and the wide type CAV-2 was found. These results indicated that this recombinant virus CAV-2-deltaE3 (NF) may be an efficient vector for gene transfer and the capacity of the vector for inserted foreign gene was up to 3.3kb.