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In this study, a novel polyurethane porous 3D scaffold based on polyethylene glycol (PEG) and polytetrahydrofuran glycol (PTMG) was developed by in situ polymerization and freeze drying. Aliphatic hexamethylene diisocyanate (HDI) as a nontoxic and safe agent was adopted to produce the rigid segment in polyurethane polymerization. The chemical structure, macrostructure, and morphology—as well as mechanical strength of the scaffolds—were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), and tensile tests. The results show that the HDI can react mildly with hydroxyl (–OH) groups of PEG and PTMG, while gas foaming action caused by the release of CO2 occurred simultaneously in the reactive process, resulting in a uniform porous structure of PU scaffold. Moreover, the scaffolds were soaked in water and freeze dried to obtain higher porosity and more interconnective microstructures. The scaffolds have a porosity of over 70% and pore size from 100 to 800 μm. The mechanical properties increased with increasing PEG content, while the hydrophilicity increased as well. After immersion in simulated body fluid (SBF), the scaffolds presented a stable surface structure. The gas foaming/freezing drying process is an excellent method to prepare skin tissue engineering scaffold from PTMG/PEG materials with high porosity and good inter connectivity. [ABSTRACT FROM AUTHOR]