Chlamydia psittaci (C. psittaci) is the causative agents of avian chlamydiosis and bovine enzootic abortion respectively. Here we describe a fluorescent quantitative real-time PCR (FQ-PCR) method developed for sensitive and specific measurement of C. psittaci infections in cattle based on TaqMan MGB primers and probes targeting the ompA gene of C. psittaci. The specificity and sensitivity of the established FQ-PCR assay were assessed. Besides, the established FQ-PCR assay was applied to detect C. psittaci in 376 specimens which collected from three large-scale dairy farms. The experimental results indicated that the detection limit for this FQ-PCR assay was 10 fg of total DNA, with a sensitivity of 100 times higher than that of the conventional gel-based semi-quantitative PCR assay targeting the ompA gene, and there was no cross-reactivity with Chlamydia trachomatis, Chlamydia pneumoniae, etc Mycobacterium bovis, Brucella, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus, Streptococcus lactis, Klebsiella pneumoniae and Salmonella, indicating that the established FQ-PCR assay had and high specificity. Furthermore, examination of field samples by the hemagglutination test kit, semi-quantitative PCR and established FQ-PCR revealed that the sensitivity and specificity of this FQ-PCR assay was significantly higher than IHA assay and PCR assay. This FQ-PCR assay for sensitive and specific detection of C. psittaci is suited for routine diagnosis, which renders it a useful tool for the recognition of outbreaks of psittacosis and bovine enzootic abortion.