The effects of transgenic Bacillus thuringiensis (Bt) cotton on non-target Aphis gossypii were assessed by comparing the life-table parameters of aphids that fed on Bt vs. non-Bt cotton cultivars for the first and 37th generations. The enzyme-linked immunosorbent assay (ELISA) method was used to detect the transmission of the Bt protein from Bt cotton to A. gossypii and their honeydew. We found that the life-table parameters of A. gossypii that fed on Bt cotton for the first and 37th generations did not differ significantly from those of the non-Bt-fed individuals. However, the Bt protein was detected by ELISA in the Bt cotton leaves, and the content varied significantly at different growth stages. Furthermore, trace amounts of the Bt protein were detected in some of the Bt-fed aphids, and the honeydew of the Bt-fed aphids contained over 10 ng/g Bt protein. These results indicate that although trace amounts of the Bt protein were ingested, the Bt cotton had no significant negative impacts on A. gossypii in either the short or long term.