Dengue infection is a global burden affecting millions of world population. Previous studies indicated that flavanones were potential dengue virus inhibitors. We discovered that a novel flavanone derivative, 5-hydroxy-7-methoxy-6-methylflavanone (FN5Y), inhibited DENV2 pH-dependent fusion in cell-based system with strong binding efficiency to DENV envelope protein at K (P83, L107, K128, L198), K' (T48, E49, A50, L198, Q200, L277), X' (Y138, V354, I357), and Y' (V97, R99, N103, K246) by molecular dynamic simulation. FN5Y inhibited DENV2 infectivity with EC₅₀s (and selectivity index) of 15.99?±?5.38 (>6.25), and 12.31?±?1.64 (2.23) μM in LLC/MK2 and Vero cell lines, respectively, and inhibited DENV4 at 11.70?±?6.04 (>8.55) μM. CC50s in LLC/MK2, HEK-293, and HepG2 cell lines at 72?h were higher than 100?μM. Time-of-addition study revealed that the maximal efficacy was achieved at early after infection corresponded with pH-dependent fusion. Inactivating the viral particle, interfering with cellular receptors, inhibiting viral protease, or the virus replication complex were not major targets of this compound. FN5Y could become a potent anti-flaviviral drug and can be structurally modified for higher potency using simulation to DENV envelope as a molecular target.