Waterborne polyurethanes (WPUs) with natural dimer fatty acids (DA) based polyester polyols as soft segments were prepared via the acetone process, and characterized by Fourier transform infrared (FTIR) spectroscopy and gel permeation chromatography (GPC). The thermal and mechanical properties were investigated using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and tensile tester. Water contact angle on the surface of the WPU films increased from 80° to 93° with increasing DA weight content in dicarboxylic acid of polyester polyol from 0 to 100%. The results indicated that among the synthesized WPUs, those containing DA exhibited excellent water resistance (water uptake decreased from 13.2% to 2.42%), outstanding hydrolytic resistance (weight loss decreased from 14.2% to 5.85%) and superior thermal stability (decomposition temperature of 50% weight loss and maximum weight loss, increased from 384 to 443°C and from 409 to 464°C, respectively). Compared to toluene resistance and mechanical properties of WPUs containing no DA, those of WPUs containing DA were low. For the WPU films based on polyester polyol derived from 100% DA as dicarboxylic acid, the interesting delamination occurred in special solvents, probably due to dissolution of low molecular weight molecules, and the presence of long hydrophobic branched chains and the high degree of phase separation.