Abstract: Dietary analysis of fish is an important part of fish biology and ecology as well as the basis of fishery resources development. In this study, the gastric DNA of Diaphus splendidus was extracted, and the mitochondrial cytochrome c oxidase subunit I (COI) was amplified as a molecular marker. The food composition was identified by high-throughput sequencing. The results show that a total of 34 species belonged to 5 phyla, 7 classes, 11 orders, 18 families and 29 genera. The diet of D. splendidus mainly included Ostracoda, Copepoda, Amphipoda, Fish, Jellyfish, Euphausiacea, Gastropod and Polychaeta. Ostracoda, Copepoda and Amphipoda were the dominant groups in its food composition, which is basically consistent with the previous research based on traditional morphological identification, except for jellyfish that was identified only by this study. In conclusion, DNA metabarcoding method is suitable for the food analysis of D. splendidus in the South China Sea, and has higher identification potential than the food analysis based on morphology.