[Objective] Bartonella bacilliformis is a fastidious haemotropic gram-negative bacterium, an etiological agent of a life-threatening illness, termed Carrion’s disease or bartonellosis. We developed a rapid, highly sensitive and specific assay, based on real-time fluorescence quantitative PCR for identifying B. bacilliformis. [Methods] A primer pair and probe set for B. bacilliformis were designed based on a species-specific gene of B. bacilliformis, based on bioinformatics method by scanning and comparing of all bacteria on GenBank data. The annealing temperature and the final concentration of the TaqMan probe and primers were optimized. The amplification product of the target gene was cloned into pEASY-T vector for preparing the standard and making a standard curve. The standard curve was made using 10×dilution series of the reference plasmid and the efficiency and linearity of PCR amplification were analyzed. Specificity, sensitivity and reproducibility of the PCR system were assessed. [Results] The optimized annealing temperature is 60 °C and the probe and primer concentration are 200 nmol/L for 20 μL reaction system. The TaqMan probe-based fluorescence quantitative PCR did not show cross reactivity with other Bartonella species, non-Bartonella bacteria and the other animals. The correlation coefficient R2 and E-value of the standard curve were 1.0 and 98.18%, respectively. [Conclusion] The real-time PCR with TaqMan-MGB probe assay is highly specific and sensitive for the detection of B. bacilliformis.