The dengue NS2B-NS3 protease existing in equilibrium between the active and inactive forms is essential for virus replication, thus representing a key drug target. Here, myricetin, a plant flavonoid, was characterized to noncompetitively inhibit the dengue protease. Further NMR study identified the protease residues perturbed by binding to myricetin, which were utilized to construct the myricetin-protease complexes. Strikingly, in the active form, myricetin binds to a new allosteric site (AS2) far away from the active site pocket and the allosteric site (AS1) for binding curcumin, while in the inactive form, it binds to both AS1 and AS2. To decipher the mechanism for the allosteric inhibition by myricetin, we conducted molecular dynamics simulations on different forms of dengue NS2B-NS3 proteases. Unexpectedly, the binding of myricetin to AS2 is sufficient to disrupt the active conformation by displacing the characteristic NS2B C-terminal β-hairpin from the active site pocket. By contrast, the binding of myricetin to AS1 and AS2 results in locking the inactive conformation. Therefore, myricetin represents the first small molecule, which allosterically inhibits the dengue protease by both disrupting the active conformation and locking the inactive conformation. The results enforce the notion that a global allosteric network exists in the dengue NS2B-NS3 protease, which is susceptible to allosteric inhibition by small molecules such as myricetin and curcumin. As myricetin has been extensively used as a food additive, it might be directly utilized to fight the dengue infections and as a promising starting material for further design of potent allosteric inhibitors.