Quantitative establishment of predator-prey food web is the foundation of effective pest management. Common methods of establishing food webs include usingmonoclonal antibodies, PCR-based gut content analysis and real-time, fluorescent, quantitative PCR. These methods are highly sensitive and species-specific, but can only qualitatively, or quantitatively, detect known prey or host species. DNA barcoding is a system of species identification using short, species-specific DNA sequences that rapidly identify species and that was first used in insect taxonomy. With the development of high-throughput sequencing technology, DNA metabarcoding based on high-throughput sequencing has a wider range of PCR amplification for prey. Unlike conventional molecular methods, this method is not limited to detecting known prey (or host) species, but can rapidly identify both known, and unknown, prey (or host) species, thereby allowing the construction of relatively complete food webs. This paper summarizes the technological roadmap of using the DNA barcode technique to establish a predator-prey food web. It also introduces the selection principles for barcode genes, design and verification methods of universal primers, as well as the basic flow of high-throughput sequencing and data analysis. The application of the method is illustrated with an example. Finally, we describe the advantages of DNA barcoding and summarize the current controversy over the use of this method. The aim of this paper is to provide theoretical guidance and support for research on the ecological regulation and management of pests based on the protection and utilization of their natural enemies, thereby enhancing the stability of food webs.