The deep sea provides global vital functions such as sequestration of carbon from the atmosphere. The increased anthropogenic pressures and interest in expanding deep-sea fisheries make this pristine ecosystem particularly vulnerable, whose conservation largely depends on rapid knowledge acquisition. In view of the limitations of traditional methods to explore the biodiversity of this vast ecosystem, the analysis of traces of macroorganismal DNA released into the water column arises as a cost-effective, noninvasive alternative. Yet, the success of this approach requires understanding of the stratification of DNA traces in the ocean. This study provides evidence that fish DNA traces can be used to establish depth-specific fish diversity and abundance throughout the water column, opening a promising avenue for gathering knowledge about the deep-sea ecosystem.Establishing the foundations for a sustainable use of deep-sea resources relies on increasing knowledge on this inaccessible ecosystem, which is challenging with traditional methods. The analysis of environmental DNA (eDNA) emerges as an alternative, but it has been scarcely applied to deep-sea fish. Here, we have analyzed the fish eDNA contained in oceanic vertical profile samples (up to 2000 m depth) collected throughout the continental slope of the Bay of Biscay. We detected 52 different fish species, of which 25 were classified as deep-sea fish. We found an increase of deep-sea fish richness and abundance with depth, and that eDNA reflects daynight community patterns and species-specific vertical distributions that are consistent with the known diel migratory behavior of many mesopelagic fishes. These findings highlight the potential of eDNA to improve knowledge on the fish species inhabiting the dark ocean before this still pristine ecosystem is further exploited.