Graphene nanoplatelets with different surface functional groups and polarity were prepared by Hummer's method and electrochemical exfoliation of graphite in two aqueous acids. The XRD, FTIR and sedimentation tests performed to characterize the polarity of the prepared graphenes. The highest polarity was associated with the sample prepared by Hummer's method and the sample synthesized by electrochemical exfoliation in nitric acid medium showed the moderate polarity. Meanwhile, the sample prepared in sulfuric acid medium showed the lowest polarity. Then thermoplastic polyurethane (TPU)/graphene nanocomposite films were fabricated with solvent exchange method. While the dispersion state and interaction of graphene/segmented polymer in solution state were evaluated with rheological measurements, optical microscope images illustrated the well dispersion of more polar graphene in TPU nanocomposite film. Moreover, the effect of graphene polarity on the phase separation and formation of hard domains with crystalline structure was extensively discussed based on DSC test results. The changes in phase separation between hard and soft segments and the formation of hard domains with specific structures greatly affected the mechanical properties of final nanocomposite films. The electrical conductivity of TPU/graphene nanocomposite film showed the role of graphene polarity nanoplatelets on the development of the conductive network in TPU matrix.