The animal diet analysis is an important research method in animal nutrition ecology, which can be used to analyze the relationship between animals and environmental factors, the relationship between predators and prey, and the diversity of animal species. In recent years, DNA metabarcoding based on next-generation sequencing technology has been widely used in many fields of ecological research, which greatly promoted the interdisciplinary development of life science. Due to the advantages of high resolution, high efficiency and relatively low sample size, diet analysis based on DNA metabarcoding has been widely applied in many prospects. Here, we summarized the principles and methods of DNA metabarcoding and diet analysis, reviewed its application in ecological research. In detail, the applications of diet analysis in biodiversity monitoring, the rare and endangered animal protection, and the agricultural pest control were summarized. In addition, the deficiencies and prospects of DNA metabarcoding in animal diet analysis were also discussed.