Tissue samples from a sheepdog infected with clinically suspected Pseudorabies virus（PRV）were identified by grinding，cell culture，subculture，CPE observation，PCR detection，virus titer determination，electron microscope observation，IFA assay，rabbit inoculation test gene sequencing and analysis. As a result，one PRV strain was isolated from dog successfully，named XJ-14 strain. The isolate was able to cause typical CPE in PK-15 cells，characterized by round，broken，cell mesh and floating. The result of PRV gE-PCR was positive，with the virus titer of 10-7.45/0.1 mL. The virus particles were round，with envelope，diameter about 150 nm under the electron microscope. The reaction with PRV fluorescent antibody was positive. After challenging the XJ-14 isolate，the rabbits appeared itching，paralysis and death. The major virulence genes，such as gE，TK and gC gene were sequenced and analyzed. The results showed that two Asp were inserted in the gE glycoprotein 48 and 497 amino acid site，respectively. And there were 12 amino acid sites mutation. For the gC glycoprotein sequence，there were 7 amino acids insertion（AASTPAA）in the 64~70 amino acid sites. All the results showed that the dog case was caused by PRV.