Abstract: A real-time fluorescence PCR method was established by using a pair of specific primers and doublefluorescence labeling probe according to the conserved sequence of the total nucleic acid extracted from the diseased jujubes causing brown spot. The real-time fluorescence PCR was used to detect other two kinds of plant disease pathogens on jujubes and six standard strains of Alternaria alternaria purchased by general microbiological culture collection center. The results showed that fluorescence signal could be collected with the combination of the specific primers and probe only in the pathogen causing brown spot on jujubes. The assay for specific detection was more sensitive than conventional PCR, which could detect the limit concentration of diseased samples as low as 1.4pg/μL analyzed by the standard curve in this method and have no need of culture, conventional identification and microscopic examination of the pathogens with the traditional method of biology. This reliable, sensitive, quick and easy-handling method could reach the species level and suitable for screening, dynamic monitoring and identification of plant diseases.