Cubozoan jellyfish pose a risk of envenomation to humans and a threat to many businesses, yet crucial gaps exist in determining threats to stakeholders and understanding their ecology. Environmental DNA (eDNA) provides a cost-effective method for detection that is less labour intensive and provides a higher probability of detection. The objective of this study was to develop, optimise and trial the use of eDNA to detect the Australian box jellyfish, Chironex fleckeri. This species was the focus of this study as it is known to have the strongest venom of any cubozoan; it is responsible for more than 200 recorded deaths in the Indo-Pacific region. Further, its ecology is poorly known. Herein, a specific and sensitive probe-based assay, multiplexed with an endogenous control assay, was developed, and successfully utilised to detect the deadly jellyfish species and differentiate them from closely related taxa. A rapid eDNA decay rate of greater than 99% within 27 h was found with no detectable influence from temperature. The robustness of the technique indicates that it will be of high utility for detection and to address knowledge gaps in the ecology of C. fleckeri; further, it has broad applicability to other types of zooplankton.