Abstract: Firstly, a series of bio-based poly(isosorbide dicarboxylate) diols (PISA) were synthesized by the reactions between bio-based isosorbide (IS) and five kinds of dicarboxylic acids with different methylene chain lengths. Then, bio-based polyurethane (Bio-PU) was prepared by the reaction between 4,4'-diphenylmethane diisocyanate (MDI) with PISA and 1,4-butanediol (BDO) as the chain extender. The structure and thermal properties of PISA samples were characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (¹H-NMR) and differential scanning calorimeter (DSC). The structure and properties of Bio-PU were characterized by FT-IR, DSC, dynamic mechanical thermal analyzer (DMA), atomic force microscope (AFM), water contact angle test and measurement of mechanical properties. Results showed that M_n of PISA were in the range between 672 and 940. As the carbon numbers in repeating units of PISA increased from 4 to 12, the glass transition temperature (T_g) of PISA samples decreased from 10.5 °C to −40.9 ℃. The viscosity also decreased, but the crystallinity increased. When the length of methylene chain in the repeating unit of PISA increased, the hydrogen bonding of Bio-PU decreased. T_g values decreased from 114 °C to 71.4 °C. The yield strength, Young's modulus and Shore D hardness decreased from 62.9, 2 042 MPa and 80 to 53.4, 1 070 MPa and 72, respectively. The hydrophilicity also decreased. However, the tensile strength and elongation at break were improved appreciably from 36.0 MPa and 46% to 64.5 MPa and 220%, respectively. A new way to prepare Bio-PU with high rigidity and mechanical properties from IS is provided.