Objective To establish an effective PCR assay for detection of Bartonella, and application of this assay in tree shrew. Methods Sequence of Bartonella was obtained from NCBI Genbank. Three pairs of primers were designed based on this sequence. One pair of primers was determined through amplifying the major strains in China. Sixty tree shrew blood samples were tested with this PCR assay. The positive amplified fragments were sequenced to verify the reliability of this method. Results A PCR method for detection of Bartonella is successfully established, with a high specificity and the sensitivity was of 2.0×10^-5 μg/mL. Among the tested 60 blood samples, 15 positive cases were detected. Sequencing of the samples confirmed a 25% infection rate of Bartonella in the tree shrews, well consistent with the amplification results, and verified the applicability of this detection method. Conclusions The establishment of this method provides the basis for detection of Bartonella in tree shrew.