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Dengue virus (DENV) is one of the most geographically distributed mosquito-borne flaviviruses, like Japanese encephalitis virus (JEV), and Zika virus (ZIKV). In this study, a library of the known and novel Glycyrrhizic acid (GL) derivatives bearing amino acid residues or their methyl/ethyl esters in the carbohydrate part were synthesized and studied as DENV inhibitors in vitro using the cytopathic effect (CPE), viral infectivity and virus yield assays with DENV1 and DENV-2 in Vero E6 and A549 cells. Among the GL conjugates tested, compound hits GL-D-ValOMe <b>3</b>, GL-TyrOMe <b>6</b>, GL-PheOEt <b>11</b>, and GL-LysOMe <b>21</b> were discovered to have better antiviral activity than GL, with IC50 values ranging from <0.1 to 5.98 μM on the in vitro infectivity of DENV1 and DENV2 in Vero E6 and A549 cells. Compound hits <b>3, 6, 11,</b> and <b>21</b> had a concentration-dependent inhibition on the virus yield in Vero E6, in which GL-D-ValOMe <b>3</b> and GL-PheOEt <b>11</b> were the most active inhibitors of DENV2 yield. Meanwhile, the time-of-addition assay indicated that conjugates GL-D-ValOMe <b>3</b> and GL-PheOEt <b>11</b> exhibited a substantial decrease in the DENV2 attachment stage. Subsequently, chimeric single-round infectious particles (SRIPs) of DENV2 C-prM-E protein/JEV replicon and DENV2 prM-E/ZIKV replicon were utilized for the DENV envelope I protein-mediated attachment assay. GL conjugates <b>3</b> and <b>11</b> significantly reduced the attachment of chimeric DENV2 C-prM-E/JEV and DENV2 prM-E/ZIKV SRIPs onto Vero E6 cells in a concentration-dependent manner but did not impede the attachment of wild-type JEV CprME/JEV and ZIKV prM-E/ZIKV SRIPs, indicating the inhibition of Compounds <b>3</b> and <b>11</b> on DENV2 E-mediated attachment<b>.</b> Molecular docking data revealed that Compounds <b>3</b> and <b>11</b> have hydrophobic interactions within a hydrophobic pocket among the interfaces of Domains I, II, and the stem region of the DENV2 envelope (E) protein. These results displayed that Compounds <b>3</b> and <b>11</b> were the lead compounds targeting the DENV E protein. Altogether, our findings provide new insights into the structure–activity relationship of GL derivatives conjugated with amino acid residues and can be the new fundamental basis for the search and development of novel flavivirus inhibitors based on natural compounds.