The microphase separation of polyurethane is the result of thermodynamic incompatibility between the hard and soft segments, which plays a significant role in its micro structure and performance. In recent years, many researches about the microphase separation of polyurethane have been reported. However, there is a gap in summarizing the recent advances in the characterization methods and applications about it. To fill in this gap, this paper carefully compared and analyzed the characterization methods of the microphase separation such as Fourier transform infrared spectroscopy (FT-IR), Differential scanning calorimetry (DSC), Dynamic thermomechanical analyzer (DMA), Atomic force microscopy (AFM), Transmission electron microscopy (TEM), and Small-angle X-ray scattering (SAXS). Moreover, applicaitions of the microphase separated polyurethane in shape memory, water resistance, and gas separation are also discussed in detail.