The main purpose of this work was to analyze the effect of the number of unreacted isocyanate groups and the [NCO]/[OH] molar ratio during the chain extension of a prepolymer during the polymerization process on the structure, processing and selected properties of thermoplastic poly(ether-urethane)s. Three series of novel thermoplastic polyurethanes were obtained via a prepolymer route. Three prepolymers were synthesized from diisocyanate and bio-based polyol, which contained 6, 7 and 8% of unreacted isocyanate groups. The prepolymer chains were extended by using bio-based 1,4-butanediol with four different [NCO]/[OH] molar ratios. It was confirmed that the concentration of unreacted isocyanate groups in the prepolymer has an effect on the degree of the hard segment phase separation of poly(ether-urethane)s. It was also found that the melt flow index has a significant impact on the processing of polyurethanes which can be correlated to the chemical structure and dynamic mechanical properties of the polyurethanes.