Borna disease virus (BDV) is a non-segmented, negative-stranded RNA virus, which infects cells of the central nervous system (CNS) in many different species. BDV is the causative agent of the neurological disorders in horses and sheep termed classical Borna disease (BD), as well as staggering disease in cats. At present, the diagnosis staggering disease or feline BD is made by histopathology or immunohistochemistry of the CNS. In order to obtain a better clinical diagnostic tool, a duplex real-time RT-PCR assay (rRT-PCR) was developed. TaqMan ® probes and primers specific for the BDV P and BDV L genes were designed by aligning the sequences of known BDV strains. After optimisation, the sensitivity and specificity of the rRT-PCR were established. The detection limit was set to 10–100 viral genomic copies per reaction and the assay detects the BDV strains V and He/80, as well as the most divergent BDV strain known so far, No/98. Furthermore, the system detected feline BDV variants in five naturally infected cats and a feline isolate used in experimental infection of cats. This rRT-PCR assay will be a powerful tool in further studies of BDV, including epidemiological screening and diagnosis.