The invention discloses a clonorchiasis sinensis and angiostrongyliasis cantonensis real-time fluorescence PCR (Polymerase Chain Reaction) detection reagent, and a kit and a detection method thereof, wherein the detection reagent comprises two groups of compositions taking conserved segments of clonorchiasis sinensis ITS1 gene and angiostrongyliasis cantonensis ITS2 gene as targets, and the two groups of compositions respectively comprise a pair of specific primers, a specific probe and an internal standard probe. The kit comprises PCR mixed solution, Taq DNA polymerase, DEPC-H2O, a positive quality control product, a negative quality control product and pseudovirus internal standard solution; the PCR mixed solution contains the detection reagent. The detection method comprises the steps of total DNA extraction, reaction composition preparation, amplification and result judgment. The reagent and the kit are strong in specificity, high in sensitivity, pollution-free, high-throughput, and capable of accurately detecting parasite larvae, invisible to the human eyes, in food, and the detection method is fast, specific and sensitive and capable of simultaneously detecting a great amount of samples.