Notothenioide is the dominant species in the Southern Ocean. Due to its high degree of specialization in morphological characteristics and physiological adaptation, the small size of most species and the difficulty in obtaining samples, the traditional morphological classification method has great limitations. DNA barcode technology provides the standard of genetic taxonomy and effective molecular taxonomy, and is the improvement, development and innovation of traditional taxonomy methods, which is helpful to accurately locate the specimens that need to be re-identified in morphology. 97 Antarctic fish samples were obtained from Antarctic krill fisheries in CCAMLR 48.1, 48.2 and 48.3 sub regions. By morphological analysis and DNA barcoding, these samples were identified as 17 effective species belonging to 17 genera and 4 families of Notothenioide. Only 6 species were identified according to morphological characteristics, and the other 11 species were identified accurately according to DNA barcoding. So DNA barcoding can effectively identify species in the case of lack of morphological identification experience or incomplete sample morphology. Then, 97 DNA barcodes of Notothenioide were analyzed. It was found that the highest GC content in the first codon and the lowest GC content in the second codon were the unique phenomenon of Notothenioide. However, its biological significance needs to be further studied. A phylogenetic tree of 81 species of fishes belonging to 39 genera and 4 families was constructed by combining the DNA barcodes of 17 species collected in this study with 64 species screened by bold. This is the most abundant molecular identification and phylogenetic analysis of Notothenioide up to now. Genetic distance and phylogenetic tree analysis further proved the applicability and feasibility of DNA barcoding for species identification of Notothenioide. In addition, it was found that the molecular phylogenetic relationship and morphological taxonomy position of individual species of Notothenioide were inconsistent. The results of this study confirmed the efficiency of DNA barcoding technology in the identification of suborder taxa of Antarctic fish, made up for the limitations and shortcomings of traditional morphological identification methods, enriched the DNA barcoding database of Antarctic fish, laid the foundation for the study of Antarctic fish diversity and adaptive evolution, and provided scientific basis for the protection and sustainable development and utilization of Antarctic fishery resources.