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.