Objective To establish a rapid, specific and accurate method to identify and detect Yersinia pestis with Peptide Nucleic Acid (PNA) probes and streptavidin modified magnetic nanoparticles or Cy5-nanoparticles using fluorescence scan technology. Methods A pair of PNA probes were synthesized with Expedite 8909 nucleic acid synthesis system after being designed based on sequence of caf1 gene in plasmid pMT1 of Y. pestis, then they were biotinylated and conjugated onto streptavidin modified magnetic beads or Cy5-nanoparticles, respectively. After the hybridization between the probes and the target DNA, fluorescence scan technology was used to detect the DNA of Y. pestis. The optimal reaction conditions were definitely explored and sensitivity and specificity of the assay were evaluated. On the other hand, the performence between PNA probes and DNA probes, between magnetic nanoparticles provided by Hunan University and commercially available magnetic beads, as well as between Cy5-nanoparticles and single Cy5 molecule were investigated to validate which one has the advantages in nucleic acid hybridization field.Results The PNA probe-based method for detection of Yersinia pestis was established and optimized, the detection limit is 0.9μg/mL (target DNA). PNA probes, magnetic nanoparticles and Cy5-nanoparticles showed significant advantages over the similar products from the market.Conclusions PNA probes displayed excellent hybridization and highly specific recognization capability to target DNA due to their unique structure characteristics. Magnetic nanoparticles can separate the free nucleic acid or excessive probes from probes-target DNA complex easily and effectively, which improved the detection sensitivity. The stability for light and fluorescence signature magnification ability of Cy5-nanoparticles increased the detection sensitivity thousands of times compared