In this paper, a set of primers (F11/R11) and probe (TaqMan-11) specific for Bursaphelenchus xylophilus was designed to target the ribosomal DNA internal transcribed spacer (ITS) region, which composed the real-time polymerase chain reaction (PCR) assay. Then, B. xylophilus, B. mucronatus, B. hofmanni, A. macronucleatus and Seinura sp. separated from dead pine were detected a using this real-time PCR assay. The results showed that fluorescent signals were detected for all B. xylophilus isolates and there was no signal for B. mucronatus, B. hofmanni, Aphelenchoides macronucleatus, Seinura sp. and water (control). This indicated that the probe (TaqMan-11) and primers (F11/R11) were highly specific for B. xylophulus. Besides, the assay was very sensitive, detecting as little as 0.005 pg of B. xylophilus DNA in 10μL volume. The real-time PCR assay also successfully detected single pinewood nematodes, and it should be very useful for quarantine purposes.