Insect-resistant genetically modified (IRGM) rice is on the verge of commercial release in China, however, its potential non-target effect on non-target insect natural enemies remains controversial. Tracking trophic interactions between predators and preys in IRGM rice ecosystem can provide new insights into better understanding of the ecological risks of IRGM rice. In the present study, a novel method based on ligase detection reaction (LDR), PCR-LDR was introduced to track 15 prey species in the gut of a predaceous spider Pirata subpiraticus, a dominant natural enemy in rice field. Our results indicated that PCR-LDR could provide high specificity and sensitivity in tracking prey-predator interactions in rice ecosystems. PCR-LDR could detect as little as 1,000th of DNA mixture. Reliable detection of DNA samples of prey species using PCR-LDR could be significantly affected by digestion time and prey species. In the analysis of 200 field-collected P. subpiraticus and 105 field-collected Tetragnatha maxillosa individuals using PCR-LDR, prey remains were identified in 78.3 and 74.3% of the individuals, respectively, from which significant predation differences between the two spider species were observed. Predation behavior of the spider species was not significantly different between Bt and non-Bt control rice lines. These results indicated that PCR-LDR can be used as an important tool for ecological studies, especially on the interactions between predators and preys in IRGM rice or other similar ecosystems.