Abstract: In order to accurately and rapidly detect the quarantine fungi Diaporthe caulivora,a species-specific real-time polymerase chain reaction（RT-PCR） assay was developed for the detection of D. caulivora. A pair of specific primers and a TaqMan-MGB probe were designed and synthesized according to the difference of ITS sequence between D. caulivora type and related species. A novel real-time fluorescent PCR was established to detect D. caulivora. The minimal detectable concentration of targeted DNA was 1.0 pg in 10 μL reaction mixture. Optimal primer concentration and probe concentration were 0.5 μmol·L^-1 and 0.6 μmol·L^-1,respectively. The method could be used for the detection and preliminary screening of the samples suspected of carrying D. caulivora. The new method is more accurate, sensitive and time-saving than the traditional method and is suitable for routine use. It also provides a valuable tool for early rapid sensitive detection and identification of D. caulivora in plants and gives plant protection offices the chance to react accordingly and to evaluate measures for plant disease management against D. caulivora.