Lithium metal is a promising anode candidate for the next-generation higher energy density batteries. In this work, a detailed study is carried out to clearly explore the influence of separator wettability on ionic conduction and interface properties for lithium-metal anode batteries. Firstly, polyphenol and polyamine are facilely assembled on the surface of polyethylene (PE) separator with the assistant of periodate. Wettability and electrolyte uptake of the polyphenol-polyamine treated PE separator improves significantly, which resulted in the increase of ionic conductivity and lithium-ion transference number (from 0.37 to 0.49). Subsequently, galvanostatic measurements and electrochemical impedance spectra (EIS) are performed on Li symmetric cells to investigate the effect of separator wettability on interface properties. It is found that the modified PE separator favors the electrochemical process by providing lower interfacial resistance, better interface compatibility, and more uniform deposition of Li+, which correspondingly mitigate the formation of Li dendrites. Finally, lithium-metal anode cells (LiCoO2 (LCO)/Li) assembled with different separators are tested, and superior battery performance is displayed in case of the polyphenol-polyamine treated substrate. These results are expected to be instructive for the design of more durable Li electrodes.