The food composition of Gymnocypris eckloni, an indigenous fish species of Eling Lake at the source of the Yellow River, can lay a foundation for its biology. In the present study, feeding intensity, food composition, feeding habits, and their changes with the growth of Gymnocypris eckloni collected in June 2023 from Eling Lake were studied and analyzed by using microscopic analysis and DNA metabarcoding. A total of 7 taxa and 26 primary dietary groups were identified from the intestinal contents of 22 Gymnocypris eckloni using microscopic analysis. Metabarcoding technology detected 12 phyla and 43 genera of bait organisms. The food diversity index value and food species varied with the increase in size of Gymnocypris eckloni, and the dominant dietary group was Cnidaria. The diversity of bait organisms in the intestinal contents was higher for large Gymnocypris eckloni individuals than for medium-sized and small-sized Gymnocypris eckloni individuals. The present study, combining microscopic analysis and DNA metabarcoding, has greatly enhanced the ability to characterize the dietary habits of the Gymnocypris eckloni. This study provides important basic data and information for conducting artificial culture and determining the nutritional requirements and bait feeding and breeding characteristics of Gymnocypris eckloni.