In order to determine the transference of Bacillus thuringiensis Berliner (Bacillaceae) (Bt) insecticidal protein in the food chain, enzyme-linked immunosorbent assay was used to detect Bt insecticidal protein levels in transgenic Bt cotton (GK12, New variety 33B and SGK321), Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) fed on the Bt cotton varieties, and two natural enemies of S. exigua, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) and Microplitis pallidipes Szépligeti (Hymenoptera: Braconidae). The results showed that Bt insecticidal protein was found not only in cotton leaves, but also in the body and excrement of S. exigua and the bodies of both C. carnea and M. pallidipes. Bt toxin was detected in S. exigua larvae of all the examined instars (second, third, fourth, and fifth) that fed on transgenic cotton varieties and the Bt toxin level was the highest in the body of the second instar. In addition, the Bt toxin content in the excrement of the second instar was lower than that in the older ones. After the natural enemies C. carnea and M. pallidipes preyed/parasitised the S. exigua larvae that fed on transgenic cotton, Bt toxin was found in both the predator and parasite. This research indicates that Bt protein can be transferred through the food chain and to natural enemies of various predatory habits.