Dengue virus (DENV) infection has become a public health problem worldwide. The development of anti-DENV drug is urgently needed because neither licensed vaccine nor specific drug is currently available. Inhibition of DENV attachment and entry to host cells by blocking DENV envelope (E) protein is an attractive strategy for anti-DENV drug development. A hydrophobic pocket on the DENV E protein is essential for structural transition in the membrane fusion, and inhibition of this process is able to inhibit DENV infection. To search for a safe anti-DENV drug, we identified short peptides targeting the hydrophobic pocket by molecular docking. In addition, the information of predicted ligand-binding site of reported active compounds of DENV2 hydrophobic pocket was also used for peptide inhibitors selection. The di-peptide, EF, was the most effective on DENV2 infection inhibition in vitro with a half maximal inhibition concentration (IC50) of 96 μm. Treatment of DENV2 with EF at the concentration of 200 μm resulted in 83.47% and 84.15% reduction in viral genome and intracellular E protein, respectively. Among four DENV serotypes, DENV2 was the most effective for the inhibition. Our results provide the proof of concept for the development of therapeutic peptide inhibitors against DENV infection by the computer-aided molecular design.