The three sympatric angel shark species occurring in the Mediterranean – Squatina squatina (the angelshark), Squatina aculeata (the sawback angelshark), and Squatina oculata (the smoothback angelshark) – are all classed as Critically Endangered on the International Union for Conservation of Nature (IUCN) Red List. There is a clear need to better quantify their current status, using appropriate non-destructive methods, to help inform future conservation measures. This study introduces an environmental DNA (eDNA) assay able to detect and distinguish S. aculeata, S. oculata, and S. squatina in the Mediterranean Sea by combining probe-based quantitative polymerase chain reaction (qPCR) technology and Sanger sequencing. The assay targets a 173-bp barcode in the mitochondrial cytochrome c oxidase I (COI) gene. It was tested in silico, in vitro on tissue-extracted DNA, and on eDNA extracted from filtration samples. This genus-specific assay was applied to detect the presence of S. squatina in eDNA samples collected in Corsica, France. The target barcode was found in seven of 76 eDNA samples, revealing the presence of S. squatina in north-western Corsica, where the shark has never been observed, and confirming its existence on the eastern coast. The study also demonstrates that using eDNA sampling, based on 30 L of seawater filtered close to the substrate with a waterproof peristaltic pump, it was possible to detect the eDNA of this rare benthic species. The results of detection can help identify critical areas for angel shark conservation and facilitate the development of local public awareness initiatives. This novel qPCR assay should be used for future applications in the Mediterranean and eastern Atlantic targeting angel sharks to better identify the remaining populations. In this study the qPCR assay was applied for S. squatina eDNA, but application to S. aculeata and S. oculata still needs to be validated in the field.