Transient expression assays were carried out to assess the transcriptional enhancement from the immediate-early gene ( ie-1 ) promoters by cis -linked Bombyx mori nucleopolyhedrovirus (BmNPV) homologous region-3 ( hr3 ). The ie-1 promoters were derived from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and BmNPV. Hr3 was placed downstream of the luciferase ( luc ) gene, the resulting plasmids were used to transfect the Spodoptera frugiperda (Sf-21), Bombyx mori (Bm-5, Bm-N) cell lines and 5th instar silkworm larvae mediated by lipofectin. The results revealed that hr3 could stimulate the transcription of homologous BmNPV ie-1 promoter 1970, 2421, 683, and 1059 folds, and the heterologous AcMNPV ie-1 promoter 6462, 4046, 6980, and 605 folds, respectively. These results implied that hr3 was a super enhancer which could function both in vitro and in vivo. The 30-bp imperfect palindrome in hr3 was proved to be the essential structure for enhancing function of hr3 . The utilizing of this promoter–enhancer combination in the baculovirus expression vector system (BEVS) was also carried out to further confirm the function of hr3 enhancer.