Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. The NS2B-NS3 viral protease cleaves the viral polyprotein. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. Moreover, while presence of NS2B/NS3 cleavage inhibits the activity of unlinked ZIKV NS2B-NS3 protease, no inhibition of protease activity was observed for unlinked DENV NS2B-NS3 full length with NS2B/NS3 cleavage site. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of flexible NS2B and NS3 N- and C-termini. We identified a second cis-cleavage site prior to NS2B C terminus and captured the crystal structure of self-cleaved NS2B47NS3 protease in post cleavage state. We report that the cleaved NS2B C-terminus occupy the neighbouring NS2B-NS3 molecule identifying a different mechanism where DENV NS2B C-terminus modulates NS2B-NS3 protease activity via trans-interaction in contrast to the cis-interacting NS2B C-terminus from the unlinked ZIKV protease. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the solvent-exposed beta-barrel of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B.