Pseudorabies (porcine pseudorabies) is an acute infectious disease, which caused by pseudorabies virus (PRV). Clinical features of infected pigs was temperature rises, newborn piglets nerve symptom and affects the digestive system. Adult pig often hidden infection, pregnant pigs can cause abortion, stillbirth and respiratory clinical symptom, no itching. The clinical symptom of boar was reproductive failure and respiratory. This disease may also occur in other domestic and wild animal. The virulence of PRV was coordinated control by several genes, mainly gE, gD, gI and TK genes. At present the main diagnosis methods of PRV are PCR, gE-ELISA, gG-ELISA, gC-ELISA, gE-LAT (latex agglutination test) and gG-LAT etc. PCR has the advantages of fast, sensitive, specificity, can simultaneously detect large quantities of samples, and can be suitable for in vivo detection, suitable for clinical diagnosis. In this study, a pair of primers were designed for specific amplificationaccording to the PRV virus genome databases.The optimal PCR reaction system was determined through optimizing concentration of primers and the template concentration and PCR amplification conditions.The best amplification conditions as follows: 10×Buffer 5 ¼l, MgCl2 (15 mmol/L) 5 ¼l, dNTPs (2 mmol/L) 3 ¼l, the forword primer(2 ¼mol/L) 2 ¼l, reverse primer (2 ¼mol/L) 2 ¼l, Taq enzyme 0.3¼l (1.5U), DNA template 5 ¼l, add water to the 50 ¼l. PCR reaction conditions are as follows: 95! for 4 min, 35 cycles of 95! for 1 min, 65! for 1min and 72! for 1 min, and a final extension at 72! for 5 min. Electrophorese in 1% agarose and detect by ethidium bromide staining.This study provides a simple method for PRV clinical diagnosis, can be detected in 2 hours, can be used for clinical diagnosis and epidemiological survey of PRV.