The giant freshwater crayfish, Astacopsis gouldi Clark, 1936, is an endangered endemic freshwater species native to Northern Tasmania. Habitat loss, fishing pressure, and climate change have been identified as threatening processes. The Australian government approved a recovery plan for A. gouldi in 2017 that requires routine population surveys to assess the impact of ongoing threats and recovery actions. We developed a novel species-specific probe-based assay targeting a 219 bp fragment in the Cytochrome Oxidase 1 gene region to detect traces of A. gouldi DNA in environmental samples as a cost-effective, sensitive, and non-invasive surveillance method to assess the presence of this endangered species. We tested assay specificity against ten crayfish species commonly found in Tasmania within the Astacopsis, Cherax, Geocharax, Engaeus, and Ombrastacoides genera and determined assay sensitivity using tissue-derived genomic DNA and synthetic oligo standards designed for A. gouldi. We then tested water samples collected from aquaria and natural freshwater streams in Northern Tasmania with known occurrence of A. gouldi, as well as one site with no known A. gouldi occurrence. The probe-based assay designed in this study successfully detected A. gouldi DNA and eDNA with a 10 copies/µl limit of detection and showed no amplification of non-targeted co-existing crayfish species. We successfully detected the presence of A. gouldi eDNA in water samples from six sites with known occurrences of the species. There was no detection from the negative site. This study validates the use of eDNA-based detection of A. gouldi by real-time PCR as a non-invasive monitoring tool to assist field monitoring, assessment, and complement ongoing recovery actions to protect habitable ecosystems of A. gouldi.