High-throughput sequencing is a major breakthrough in the development of DNA sequencing technology and provides an unprecedented opportunity for modern biological scientific research, such as the DNA-based approach tracking the food chains among predators-prey or herbivores-host plants trophic interactions in ecosystems. This review illustrates and compares the principles of various types of technology, including the Roche 454, Illumina, and Ion Torrent technologies, and other recent progress. We also summarize studies that have used high-throughput sequencing technology to study interactions among generalist predators/herbivores and their prey/hosts. This review could provide new information and novel approaches to exploring molecular trophic interactions and improving our understanding of the prey/host spectrum, prey/host shift, biological control, resource allocation, conservation biology, and ecological restoration.