Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, causing severe central nerve system diseases without specific treatments. The NS2B–NS3 protease of flaviviruses mediates several cleavages on the flavivirus polyprotein, being believed to be a target for antiviral therapy. NS2B is the cofactor of the viral serine protease, correlating with stabilization and substrate recognition of the NS3 protease. In this study, we investigate the functional determinants in the JEV NS2B for the activation of the NS3 protease. Cis- and trans-cleavage assays of the deletions at the N-terminal of NS2B demonstrated that the NS2B residues Ser46 to Ile60 were the essential region required for both cis and trans activity of the NS3 protease. In addition, alanine substitution at the residues Trp53, Glu55, and Arg56 in NS2B significantly reduced the cis- and trans-cleavage activities of the NS3 protease. Sequence alignment and modeled structures suggested that functional determinants at the JEV NS2B residues Ser46 to Ile60, particularly in Trp53, Glu55 and Arg56 could play an important configuration required for the activity of the flavivirus NS3 protease. Our results might be useful for development of inhibitors that block the interaction between NS2B and NS3.