Waterborne polyurethane (WPU) with different hard segment content (HSC) was synthesized using PTMG-2000, IPDI , EG, DMPA and EDA as raw materials. The quantitative effects of HSC on the degree of hydrogen bonding was established by decomposition of C=O and N-H stretching of FTIR. The effect of hard segment content on microcrystalline degree and microphase separation degree were studied by DSC and XRD. The results showed that the degree of hydrogen bonding, hard segment microcrystalline degrees and microphase separation degree of WPU increased with HSC increasing.