Diarrhoea is one of the most common conditions requiring veterinary intervention in foals and is a significant economic burden on horse industry in Australia. Along with several other infectious causes of equine diarrhoea, equine adenovirus 2 (EAdV-2) has been associated with gastrointestinal infections. Sensitive detection of equine adenovirus-2 is essential in the diagnosis of this virus and differentiation from adenovirus virus-1. In this study, a quantitative polymerase chain reaction (qPCR) assay was developed to amplify a 109 bp fragment of EAdV-2 hexon gene using SYBR Green for detection and specific identification EAdV-2 nucleic acid. The standard curves generated using serial dilutions of the cloned hexon gene demonstrated a broad dynamic range between 2.7 x 10⁸ and 2.7 x 10¹ copies per reaction with a correlation coefficient (R²) of 0.994 and 106.0% efficiency. The assay had a high reproducibility with low intra and inter-assay variations at all dilutions. The analytical specificity of the assay was confirmed using a range of equine viruses (equine adenovirus-1, equine herpesvirus-5, equine rhinitis virus B1) and no specific amplification was observed. The qPCR assay can detect 10 fold less starting DNA than conventional PCR. Overall, the qPCR developed is highly sensitive, reproducible and specific. This assay will be useful in ultimately enhancing our understanding of EAdV-2 infection as well as simplifying the detection of this virus.