Prunus necrotic ringspot virus (PNRSV) is considered a quarantine pathogen of fruit trees disease in some parts of the world, and also a plant quarantine virus issued by Chinese government. A pair of primer and a TaqMan probe based on the conserved nucleotide sequence of coat protein gene of different PNRSV strains were designed and synthesized. Then through optimizing the concentration of primers, probe, Mg2+ and dNTPs, a real-time fluorescent RT-PCR was developed for detection of PNRSV in fruit trees. when optimal concentrations of primers, probe, Mg2+ and dNTPs was 400 nmol/L, 333 nmol/L, 5 mmol/L and 0.43 mmol/L, the assay for specific detection was highly sensitive, which could detect the template concentration as low as 23 copies, and could detect PNRSV in leaves tissues of cherry trees successfully. This reliable, sensitive, quick and easy-handling method is suitable for detection and identification of Prunus necrotic ringspot virus.