Fram Strait, the gateway between the Arctic and Atlantic Oceans, is undergoing major climate change-induced physical and biological transformations. In particular, rapid warming and ongoing “Atlantification” are driving species range shifts and altering food web structures in the Arctic. Understanding and predicting the consequences of these processes on future ecosystems requires detailed assessments of local and pelagic biodiversity. Gelatinous zooplankton (GZP) is an important component of pelagic communities, and recent evidence indicates that such communities are undergoing major changes in the Fram Strait. However, as sampling GZP is challenging, they are regularly underestimated in biodiversity, distribution, and abundance. To overcome this and address existing ecological knowledge gaps, we investigated patterns of pelagic metazoan diversity in Fram Strait using environmental DNA (eDNA) metabarcoding of the cytochrome c oxidase I (COI) gene. We successfully detected a broad range of taxa from the marine metazoan and GZP communities across sampling locations and ocean depth zones. We demonstrate the vertical structuring of diversity and elucidate relationships between taxa and water mass indicators, such as salinity and temperature. Furthermore, when comparing eDNA data with net and video transect data for GZP at the same period and location, we found that eDNA uncovered a higher number of taxa, including several that were not detected by the other methods. This study is a contribution to the formation of baseline Arctic GZP biodiversity datasets, as well as future research on changing marine metazoan biodiversity and community composition.