Bio-based polyester polyol was synthesized via esterification between azelaic acid and isosorbide. After esterification, bio-based polyurethanes were synthesized using polyester polyol, 1,3-propanediol as the chain extender, and 4,4'-diphenylmethane diisocyanate, in mixing ratios of 1:1:1.5, 1:1:1.8, 1:1:2, and 1:1:2.3. The bio TPU (Thermoplastic Polyurethane) samples were characterized by using FT-IR (Fourier Transform Infrared Spectroscopy), TGA (Thermal Gravimetric Analysis), DSC (Differential Scanning Calorimetry), and GPC (Gel Permeation Chromatography). The mechanical properties (tensile stress and hardness) were obtained by using UTM, a Shore A tester, and a Taber abrasion tester. The viscoelastic properties were tested by an Rubber Processing Analyzer in dynamic strain sweep and dynamic frequency test modes. The chemical resistance was tested with methanol by using the swelling test method. Based on these results, the bio TPU synthesized with the ratio of 1:1:2.3, referred to as TPU 4, showed the highest thermal decomposition temperature, the largest molecular weight, and most compact matrix structure due to the highest ratio of the hard segment in the molecular structure. It also presented the highest tensile strength, the largest elongation, and the best viscoelastic properties among the different bio TPUs synthesized herein.