The misuse of chemical pesticides and large-scale planting of monocultures have significantly reduced biodiversity in agricultural ecosystems. Strengthening the management of non-crop habitats can effectively increase the number and variety of natural enemies of insect pests, promote the establishment and stability of food webs, and thereby fully utilize the pest control functions of natural enemies within the food web. Therefore, understanding the predatory relationships and control capabilities of predatory natural enemies in the food web is a prerequisite for fully utilizing their pest control function. This paper systematically reviews progress in research methods based on DNA molecular detection technology, with a focus on evaluating the pest control capabilities of natural enemies in the food web through the analysis of their gut contents. It highlights the similarities and differences between traditional methods of detecting gut contents, and molecular detection technologies (such as PCR and DNA barcoding) in terms of sensitivity, accuracy, and operability. Furthermore, in combination with high-throughput sequencing technology, the potential of constructing food web relationships based on the cytochrome c oxidaseⅠgene in mitochondrial DNA is discussed, noting existing limitations in terms of applicability. In the future, combining quantitative predation assessments with food web construction will further reveal the potential application of DNA molecular detection technologies in biological pest control, and their innovative significance in pest ecological regulation. This will promote the deep integration of genomics and ecology, provide important scientific evidence for optimizing the pest control functions of predatory species and promote sustainable pest management in agricultural ecosystems.