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The Southern Ocean surrounding Antarctica harbours some of the most pristine marine environments remaining, but is increasingly vulnerable to anthropogenic pressures, climate change, and invasion by non-native species. Monitoring biotic responses to cumulative impacts requires temporal and spatial baselines and ongoing monitoring - traditionally, this has been obtained by continuous plankton recorder (CPR) surveys. Here, we conduct one of the longest environmental DNA (eDNA) transects yet, spanning over 3000 nautical miles from Hobart (Australia) to Davis Station (Antarctica). We evaluate eDNA sampling strategies for long-term open ocean biomonitoring by comparing two water volume and filter pore size combinations: large (12 l with 20 μm) and small (2 l with 0.45 μm). Employing a broad COI metabarcoding assay, we found the large sample/pore combination was better suited to open ocean monitoring, detecting more target DNA and rare or low abundance species. Comparisons with four simultaneously conducted CPR transects revealed that eDNA detections were more diverse than CPR, with 7 (4 unique) and 4 (1 unique) phyla detections respectively. While both methods effectively delineated biodiversity patterns across the Southern Ocean, eDNA enables surveys in the presence of sea-ice where CPR cannot be conducted. Accordingly, 16 species of concern were detected along the transect using eDNA, notably in the Antarctic region (south of 60°S). These were largely attributed to hull biofouling, a recognized pathway for marine introductions into Antarctica. Given the vulnerability of Antarctic environments to potential introductions in a warming Southern Ocean, this work underscores the importance of continued biosecurity vigilance. We advocate integrating eDNA metabarcoding with long-term CPR surveys in the Southern Ocean, emphasising the urgency of its implementation. We anticipate temporal and spatial interweaving of CPR, eDNA, and biophysical data will generate a more nuanced picture of Southern Ocean ecosystems, with significant implications for the conservation and preservation of Antarctic ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)