Environmental DNA (eDNA) is a relatively new tool for the detection of rare, threatened and invasive species in water bodies. In this study we investigated the utility of an eDNA approach in detecting the Critically Endangered largetooth sawfish Pristis pristis in freshwater habitats in northern Australia. Water samples were collected from large aquaria mesocosms containing largetooth sawfish and other aquatic species, and floodplain waterholes and the main river channel of the Daly River, Northern Territory. Water samples were filtered using a 20 µm nylon filter. DNA was extracted from filters and analysed with PCR using species-specific mitochondrial cytochrome c oxidase subunit I (COI) primers designed to amplify only largetooth sawfish DNA. PCR products were cleaned and the COI gene sequenced to confirm the species identity. Using 3 aquaria, with one containing a largetooth sawfish, this method positively identified sawfish only in the correct aquarium. In the field water samples, 7 of 8 floodplain waterholes produced a sawfish eDNA PCR product, while eDNA was not detected in the main river channel. Based on gillnet sampling and traditional ecological knowledge, largetooth sawfish were known to occur at half of the waterhole and floodplain sites that tested positive for sawfish eDNA. These results demonstrated that an eDNA approach to detecting largetooth sawfish can produce reliable outcomes and can be used as a survey tool to help with conservation efforts for this and other threatened elasmobranchs.