Abstract: The objective of this study was to develop a Real-time RT-PCR assay of chicken Infectious Bronchitis Virus (IBV) and therefore to detect the load IBV quantitatively. A fragment of N gene in IBV was amplified with RT-PCR method, and was cloned into the vector pEASY-T3. Then, the recombinant plasmid containing the N gene fragment was constructed. The standard curve and corresponding linear regression equation of IBV nucleic acid level were developed by Real-time PCR based on SYBR Green I with the recombinant plasmid. This method showed a high specificity and had a detection limit of 5.58×102 copies/μL, and its coefficient of variations was less than 3.2% in the reproducible assays. The virus nucleic acid in tissue samples from chickens inoculated experimentally with IBV M41 strain was quantitatively determined with the established Real-time RT-PCR. The detection results showed that the load of IBV in the kidney was more than that in bronchus and lung after inoculation, and the virus load in the bronchus and lung on 3 days post-inoculation (DPI) were higher than those on 7 DPI and 10 DPI. Moreover, the correlation between the clinical manifestations and viral load was confirmed. The results indicated that this Real-time RT-PCR assay was of high specificity, sensitivity and reproducibility, and could be used for the quantitative detection of IBV.