The blind cave eel, Ophisternon candidum (Mees in J R Soc West Aust 45: 24–32,1962), is a rare groundwater inhabitant found in geographically isolated populations of north-west Australia. The species is listed as vulnerable under Commonwealth legislation and is a priority consideration when environmental disturbance by resource companies is proposed. Detection of this species for Environmental Impact Assessment (EIA) and monitoring is difficult because individuals are naturally rare or traditional sampling techniques are ineffective. To properly manage the species, information on population distribution and connectivity is critical. We sought to examine whether environmental DNA (eDNA) of O. candidum could be detected and whether positive detection was correlated with previous locations where the species had been physically caught. We developed new eDNA species-specific PCR assays to screen groundwater sampling points and we detected O. candidum DNA in three boreholes where the species has previously been collected and five additional groundwater sampling points. Our results demonstrated that the newly designed assays were effective for detecting this rare and vulnerable subterranean species. This work sets a benchmark for the application of eDNA species-specific PCR assays for EIA and monitoring, and has potential for these assays to be expanded more broadly to high-throughput eDNA metabarcoding for subterranean groundwater communities in the future.