Objective To develop a rapid, sensitive and specific assay based on TaqMan probe real-time PCR to quantitate Helicobacter bilis(H.bilis). Method A 435 bp specific fragment of H.bilis P17 gene was amplified by PCR, then cloned into pMD19-T vector to construct a recombinant plasmid pMD-HBP17, which was used as standard DNA of this qPCR method. The qPCR system was optimized by using serial dilution of standard plasmid. The sensitivity, specificity, repeatability and quantitation range of this method were evaluated. The established method was used to detect 77 clinical samples. Result The quantitative standard curve from 10⁸ copies/ well to 10¹ copies/well of serial diluted plasmid DNAs showed that they had good linear correlation, the slope of the standard curve was -3.46, R²>0.999, and the lowest limit reached 2×10¹ copies/well. The positive rate of H.bilis detected by qPCR was 14.3% which is higher than detected by PCR(7.8%). Conclusion ThisqPCR method showed high sensitivity, specificity and stability and will be utilized for qualitative and quantitative detection of H.bilis.