Black rot of grape caused by Phyllosticta ampelicida (Guignardia bidwellii) is a destructive disease present in most grape growing regions worldwide, with the exception of Australia and New Zealand. P. ampelicida is a quarantine organism for New Zealand and it is important to have a fast and accurate diagnostic method for the early detection of this fungus. In this study, a real-time PCR (qPCR) assay was developed and validated for the detection of P. ampelicida. Multiple sets of P. ampelicida specific primers and probes were designed using internal transcribed spacer (ITS) DNA sequences. Optimisation experiments determined the best performing primers (Gb-IT4F and Gb-IT5R) and TaqMan probe (Gb-IT2P). The specificity of the qPCR assay was confirmed by testing against strains of P. ampelicida from Vitis sp., P. bidwellii from Parthenocissus sp., closely related Guignardia species, and other fungal isolates commonly associated with grapevines. The qPCR assay consistently detected P. ampelicida at genomic DNA concentration of 116 fg and therefore enables disease detection at low infection levels. The P. ampelicida qPCR assay was further developed as a duplex assay by co-amplifying the cytochrome oxidase (Cox) gene from plants for use as an internal PCR control. To our knowledge, this is the first qPCR developed for the rapid detection of P. ampelicida. This qPCR has the potential for use in screening imported nursery stocks and for the early detection of the pathogen during surveillance.