The cotton whitefly, Bemisia tabaci (Gennadius) B-biotype, is fed on by a wide variety of generalist predators, but there is little information on these predator–prey interactions, especially under field conditions. In this study, a real-time polymerase chain reaction (PCR) assay was developed to quantify B. tabaci B-biotype remains in predator gut. The B. tabaci B-biotype genomic DNA copy number was referred to the actual amount of BT1 isolate, the B. tabaci B-biotype specific DNA fragment. The numbers of BT1 isolate in one B. tabaci B-biotype egg, individual adult and a single red-eyed nymph were 2.56 × 103, 2.56 × 104, and 1.29 × 104 copies, respectively. When Propylaea japonica adults fed on one, two, four, eight or 16 red-eyed nymphs, the detected numbers of BT1 isolate ranged from 2.77 × 104 to 4.05 × 105 copies, forming a strong linear relationship (R2 = 0.9899). Following the consumption of two red-eyed nymphs, prey DNA was detectable in 100% of P. japonica at t = 0, decreasing to 80.0% and 60.0% after 1–4 h and 8 h of digestion, respectively, with 3.36 × 104–1.25 × 103 BT1 isolate copies. The predation by field-collected predators, 26 larvae of P. japonica, and of Harmonia axyridis each, Chrysopa spp. larvae (Chrysopa pallens and C. formosa, 18 individuals in total), and a single adult of Scymnus hoffmanni, 19 adults of Orius sauteri and nine adult spiders (Erigonnidium graminicolum and Neoscona doenitzi), on B. tabaci B-biotype were quantified. Of the 99 analysed predator individuals, 3.65 × 102–4.60 × 105 copies of BT1 isolate, equivalent to 0.8–18.8 red-eyed nymphs were detected. These results suggest that TaqMan real-time PCR technology may provide a rapid and sensitive method for quantifying B. tabaci B-biotype remains in predator guts and will be invaluable in assessing the food web relationship between prey and arthropod predators.