The Southern Ocean is experiencing complex climate change, and the Amundsen Sea is one of the regions that has responded most rapidly to climate change. Due to their role in ecosystems, environmental sensitivity and high endemism, Antarctic demersal fish are a favorable group that can act as an indicator of the response of Antarctic organisms to climate change. However, our knowledge of Antarctic fish fauna is insufficient, with knowledge gaps even in their taxonomy. This situation is greatly influenced by the limitations of traditional taxonomy and thus calls for alternative solutions such as DNA barcoding. In this study, DNA barcoding analyses of 69 fish samples obtained from the Amundsen Sea were conducted using the mitochondrial COI gene. Based on the molecular species delimitation results, 13 fish species were found to belong to two orders, six families, and 12 genera. Both the maximum likelihood and Bayesian inference methods showed that the phylogenetic relationships of Bathydraconidae were paraphyletic, which was consistent with previous phylogenetic research. Our research showed that the COI gene, as a DNA barcode, is not only suitable for the identification of Antarctic fish species but also reflects some phylogenetic characteristics that might provide important evidence and support for studies of Antarctic fish phylogenetic relationships. In summary, our study provides an important reference for fish diversity and taxonomy in the Amundsen Sea, which may further enhance our understanding of the biodiversity, taxonomy and biogeography of fish in this area.