Abstract A highly sensitive real-time PCR method was developed in this study for reticuloendotheliosis virus (REV) detection and quantitation. The real-time PCR method, with a minimum detection limit of 10 proviral DNA copies, was 100 times more sensitive than the conventional PCR. It was also shown to be highly specific, as no positive signals were detected for other common avian DNA viruses. The coefficients of variation of intra- and inter-assay reproducibility were both less than 2%. The chicken β-actin gene was co-amplified and used as the internal control to monitor the efficiency of DNA extraction and PCR amplification. Specific pathogen free chickens were infected with REV at different ages and the blood was detected with the real-time PCR method. High levels of proviral DNA were detected in the blood of REV-infected chickens during the experiment and chickens infected early had higher proviral loads from 2 weeks post-infection compared with late infected chickens. This study provides an excellent research and diagnostic tool that can be used for REV detection and quantitation.