After the closure of fishing in the Yangtze River, the recovery of carnivorous fish resources in Lake Poyang has been significant, and exploring their dietary composition in the natural environment is crucial for predicting the development tendency of fish resources in Lake Poyang. This study collected 240 specimens of Silurus asotus, Channa argus, Siniperca chuatsi, and Culter alburnus with three-layer drift gillnet at five sampling sites (Duchang, Nanjishan, Poyang, Yugan and Jinxian) in Lake Poyang from August to November 2022. The dietary composition of four fierce fish species mentioned above was analyzed using stomach content analysis and eDNA Metabarcoding technology. The results of Stomach content showed that these fish species had a high feeding rate and a low empty stomach rate. Fish constituted a significant part of their diet, followed by shrimp. The diets of S. asotus, C. argus, S. chuatsi, and C. alburnus consisted of prey from 5 categories and 10 species, 4 categories and 15 species, 3 categories and 11 species, and 5 categories and 12 species, respectively, with the highest IRI% prey being Carassius auratus, C. auratus, Hemiculter leucisculus, and Cyprinus carpio, respectively. C. auratus, C. carpio, Misgurnus anguillicaudatus, and Macrobrachium nipponense were found in the intestinal contents of all four fierce fish species. The species composition of prey organisms based on eDNA Metabarcoding technology is similar to the results of stomach contents analysis, identifying 7, 5, 6, and 6 prey species in the gut contents of S. asotus, C. argus, S. chuatsi, and C. alburnus, respectively, with C. carpio, C. carpio, C. carpio, and Hypophthalmichthys molitrix having the highest relative abundance. Additionally, eDNA Metabarcoding technology revealed new prey species not detected in stomach content analysis, including Megalobrama amblycephala, Pseudobrama simoni, Hemiculter bleekeri, and Ancherythroculter nigrocauda. In summary, after the fishing ban in the Yangtze River, the predatory fish in Lake Poyang have an abundant food supply and a wide diet range, with fish being their primary food source, followed by shrimp. And the combination of stomach content analysis and eDNA Metabarcoding technology allows for a more comprehensive investigation of fish diets, making it an effective approach for analyzing fish feeding habits.