Abstract: Objective To investigate whether Zika virus NS3 have ATP hydrolase and DNA helicase activity. Methods Zika NS3 with His tag was expressed in E.coli BL21 and purified by Nickelagarose beads. ATP hydrolysis assay was performed to examinethe ATPase activity of NS3. The dsDNA unwinding activity of NS3 was detected according to the principle of FRET(Fluorescence Resonance Energy Transfer). NS3 G198A mutant was generated by site-directed mutagenesis and its enzymatic activity was measured subsequently. Results Zika NS3 was capable of hydrolyzing ATP in vitro(Vmax=2.76 μmol /(L?min)，Km =0.11 mmol/L).Moreover, Zika NS3 could unwind dsDNA in vitro as the fluorescence was enhanced in FRET system. G198A mutation impaired NS3 enzymatic activities, including ATP hydrolysis and dsDNA unwinding activities. Conclusions Zika NS3 has DNA helicase activity in vitro depending on ATP hydrolysis, and the 198 glycine is important for its enzymatic activity.