Cotton is one of the world’s important cash crops and occupies a significant position in agricultural production and the national economy. However, insect pests severely affect the growth, yield and quality of cotton. To ensure high and stable cotton yields, the cultivation of insect-resistant transgenic cotton via transgenic technology can not only effectively reduce the impact of chemical pesticides on crops but also exert excellent control effects against pests such as cotton bollworms. In this study, the plant expression vector pC2300-VEC harboring the target genes epsps, cry1Ac and vip3A was introduced into the genome of the recipient cotton cultivar CCRI 24 via Agrobacterium-mediated transformation. The obtained transgenic cotton plants were subjected to the identification of target genes and target traits, and the insect-resistant transgenic cotton line BrsC35 was ultimately obtained. PacBio sequencing combined with conventional molecular characterization methods was used to analyze its insertion site, copy number and other characteristics, providing a new germplasm for insect-resistant transgenic cotton.