Antarctica has attracted wide attention due to the unique biodiversity, but its remote and extreme environment also poses challenges for ecosystem monitoring. Given the ecological and economic importance of the Southern Ocean, it is imperative to explore more effective biomonitoring methods. Environmental DNA (eDNA) metabarcoding, as an efficient biomonitoring tool, can be used in some special difficult to sample environments and are gradually being used in fish research in the Southern Ocean. Here, we employed eDNA to investigate the fish species diversity and assemblages in the Southern Ocean, including the Prydz Bay (PB), Cosmonaut Sea (CS), east Antarctic Peninsula (AP) and Amundsen-Bellingshausen Sea (ABS). A total of 40 species, belonging to 7 orders, 12 families and 29 genera, were detected from 121 eDNA samples in 65 stations, with 30, 31, 26 and 31 species were detected in the PB, CS, AP and ABS, respectively. There were significant differences in fish assemblages among the four areas, and the highest alpha diversity was found in the AP, followed by the ABS, CS and PB. 
Cluster analysis showed the Gunnerus Ridge of the CS had a unique species composition. Horizontal distribution showed fish species richness and alpha diversity were higher closer to the land. Comparison of eDNA results between different layers showed that species richness and alpha diversity were highest in surface and bottom layers, and that priority should be given to collecting surface water, followed by bottom water, in case of sampling difficulties. Our study demonstrates that eDNA provides insights into fish community dynamics over a wider range in the Southern Ocean.