To reveal the multi-generation effects of transgenic Bt cotton on the metabolism of secondary target herbivores, the activities of digestive and detoxification enzymes in beet armyworm, Spodoptera exigua (Hübner), fed with transgenic Bt (cv. GK-12) vs. non-Bt cotton (cv. Simian-3) for three successive generations were examined. Significantly lower activities of lipase, carboxylesterase (CarE) and acetylcholinesterase (AChE), and higher activities of trypsin and total superoxide dismutase (T-SOD) were observed in Bt-fed S. exigua than in non-Bt fed individuals during the three generations. But the activity of amylase did not differ between the Bt-fed and non-Bt fed S. exigua. Among the three generations of Bt-fed S. exigua, the activities of lipase and trypsin decreased significantly in the second or third generation than in the first generation. But those of CarE and AChE were increased significantly. Among the three generations of non-Bt fed S. exigua, the activity of lipase was significantly higher in the second and third generations than in the first generation. The increasing adaptation and fitness of S. exigua after continuous exposure to Bt cotton was supposed to be associated with the enhanced activities of detoxification enzymes. Measuring multi-generational enzymes activities of S. exigua in response to Bt cotton can provide proofs of its adaptation development, which is suggested to be a mandatory part of risk assessment of transgenic Bt plants on secondary target herbivores.