Nowadays new energies are emerging due to the fossil fuel limitations caused by global warming and environmental pollution. Among other types of new energies, electrochemical batteries are promising alternative. In addition, lithium ion batteries attracted more attentions because of their high specific energy, good performance and long span life. This study focus on investigation of electrochemical properties of lithium ion battery with polyaniline/polyurethane (PU/PANI) nanofibers membrane separator. In this study, certain amount of polyurethane nanofibers was initially dissolve in specified amount of dimethylformamide (DMF) and then PU nanofibers were produced through electrospinning. PU nanofibers morphological structure was investigated by means of both optical and scanning electron microscopes and no significantly difference between nanofiber diameters with different ratios were observed. Furthermore, aniline was synthesized in the presence of polyurethane nanoweb at different aniline concentrations (0.005, 0.01, 0.03 and 0.04 molar in mixture of hydrochloric acid and distilled water) and its morphology was analyzed by absorption of n-butanol method and scanning electron microscope. The former method results revealed that PU/PANI membranes have %68-%91 porosity. PU/PANI nanofiber membrane have been produced by impregnating nanofiber membrane in 1 molar solution of lithium perchlorate (LiClO 4 ) for 24 hours which was provided in dimethyl carbonate/ethyl carbonate (EC/DMC) solvents. The absorption percentage of electrolyte solution results demonstrated that the electrolyte take-up was between %585 and %615 and also by increasing the aniline content in membrane, its electrolyte absorption percentage had lower reduction. Nanofiber membrane ion conductance at 25 ?C temperature was measured using electrochemical impedance spectroscopy technique. The presence of synthesized polyaniline has improved the ion conductance from 0.079 mS/cm to 1.378 mS/cm. The polymeric separator which was polymerized with 0.03 molar of aniline in hydrochloric acid and distilled water (PU/PANI S3 separator) has the best ion conductance, based on its lower mass resistance than other membranes. Results showed that after the first cycle, the current has the same manner as the first cycle after passing three cycles which shows no reduction has occurred. Also the reversibility of this separator showed that it is chemically stable. Electrochemical stability of separators was investigated via CV technique using a cell composed of anode and cathode electrodes. Cyclic voltammetric test results showed that the PU/PANI S3 polymeric separator had larger area under curve than other membranes which could be because of its higher electrical capacity of battery with this separator. The results of charging/decharging test of cells containing PU/PANI separators showed that the cell having PU/PANI S3 separator had the most decharging time which indicates its highest electrical capacity than the others in this study. Based on the results obtained from ion conductance and chemical stability of PU/PANI nanofiber membranes, these membranes have a good potential to be used as polymeric separators in ion lithium batteries. Also, the synthesized membrane with 0.03 molar of aniline had the best results of ion conductance and chemical stability in ion lithium batteries.