Environmental DNA (eDNA) analysis is emerging as an innovative tool to assess the distribution and biomass of species in aquatic environments that enables quick and non-invasive surveys compared to conventional sampling methods such as fishing, hydroacoustics, and diving observations. We applied eDNA analysis to spawning ecology research of the Japanese eel Anguilla japonica in the western North Pacific for the first time. A preliminary laboratory experiment using tank water containing eels confirmed that our specific primers and probe could identify the Japanese eel from a small amount of eDNA (about 0.5 ng per PCR reaction). During a 19-20 May 2015 ocean survey at the southern West Mariana Ridge, seawater samples were collected at 12 water depths at 9 stations in the spawning area of A. japonica. Out of 108 seawater samples, DNA was successfully amplified from 3 samples, which were collected at 250 and 400 m. According to published research on Japanese eel spawning ecology and eDNA dynamics, the eDNA positive signals obtained from the oceanic survey could have been from adult Japanese eels or from larvae from the last spawning events. Thus onboard eDNA analysis proved to be a viable approach for detecting Japanese eels in their spawning area along the southern West Mariana Ridge.