Bio-based isosorbide (ISO), 1,3-propanediol (PDO) and dicarboxylic acids with various carbon chain lengths were employed to synthesize a range of isosorbide-based polyester polyols with a constant molecular weight. Then these polyester polyols were used to prepare polyurethane reactive hot-melt adhesives (PURHMAs). The thermal properties and morphology of polyester polyols were characterized by differential scanning calorimetry (DSC) and polarizing microscope (POM), respectively. The bonding properties of PURHMAs on polycarbonate (PC), iron (Fe) and aluminum (Al) were tested, and Fourier-transform infrared spectroscopy (FTIR) was used to monitor the curing process of the adhesives. Tensile test, hardness test and thermogravimetric analysis were also carried out. The impacts of isosorbide content and the carbon chain length on the properties of both polyester polyols and PURHMAs were studied in this work. The ultimate strength of PURHMAs with a longer carbon chain to PC is higher than 6.74 MPa and the highest can reach 8.04 MPa. Finally, bio-based PURHMAs prepared in this work showed good tensile properties and thermal resistance and have a very promising prospect of being applied to the adhesive industry.