Objective To develop a TaqMan MGB probe-based, sensitive and specific real-time fluorescence quantitative PCR assay for rapid detection of Cilia-associated respiratory (CAR) bacillus. Methods Primers and probes specific to 16S ribosomal RNA （16S rRNA） gene sequence of Cilia-associated respiratory bacillus were designed. A TaqMan MGB probe-based, real-time fluorescence quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed. Then, the established TaqMan MGB probe real-time fluorescence quantitative PCR assay was applied to detect Cilia-associated respiratory bacillus in 1344 clinical specimens during 2008-2012, compared with conventional PCR assay. Results The specificity of this established TaqMan MGB probe-based real-time fluorescence quantitative PCR was high for detecting Cilia-associated respiratory bacillus, and there were no cross-reactivity with Mycoplasma pneumoniae, Pasteurella pneumotropica, Bordetella bronchiseptica, Klebsiella pneumoniae, Escherichia coli and Streptococcus pneumoniae. The detection limits was 2.7 copies. The correlation coefficient and slope value of standard curve were 0.999 and -3.22, respectively and the efficiency of TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was 104.432%. The TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was preformed to detect Cilia-associated respiratory bacillus in 1344 clinical specimens, a total of 510 specimens were positive for Cilia-associated respiratory bacillus. TaqMan MGB-based probe real-time fluorescence quantitative PCR could detect Cilia-associated respiratory bacillus DNA from clinical specimens directly, and detection time is only 40 minutes. Conclusion The TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was a reliable, specific, sensitive and useful tool for rapid detection of Cilia-associated respiratory bacillus.