Abstract: In this study, azvudine (FNC), hydrochloride salt of azvudine (FNC-HCl) and triphosphate azovudine (FNC-TP) were tested against DENV-Ⅱ recombinant virus (DENV-Ⅱ Luc⁺). The inhibitory activity of FNC, FNC-HCl and FNC-TP on DENVs were detected by plaque assay. The effect on the expression of DENV-Ⅱ envelope protein E was detected by Western blot; the inhibitory of DENV-Ⅱ viral RNA by compounds was detected by real-time quantitative PCR. MTT assay was used to determine the cytotoxicity of the three compounds on Vero cells. The results showed that FNC, FNC-HCl and FNC-TP inhibited the viral replication by inhibition of renilla luciferase activity of DENV-Ⅱ Luc⁺. The 50% effective concentration (EC₅₀) of FNC, FNC-HCl and FNC-TP in the inhibition of DENVs replication were from 0.54-25.42 μmol·L^-1, while that of ribavirin was 40.78 ±1.02 μmol·L^-1 as the positive control. Western blot and real time quantitative PCR results showed that FNC, FNC-HCl and FNC-TP significantly inhibited the expression of DENV-Ⅱ E protein, and the replication of DENV-Ⅱ viral RNA. The 50% cytotoxic concentrations of FNC, FNC-HCl and FNC-TP were all greater than 3 000.00 μmol·L^-1. The results suggest that in vitro anti-DENVs activities of FNC, FNC-HCl and FNC-TP are superior to ribavirin, which are expected to become new candidates of anti-DENV drugs.