Objective To study the morphology and biomechanical properties of the improved acellularized nerve scaffold using the technique of hypotonic buffer combined with freeze-drying. Methods The traditional acellularized nerve scaffold (traditional group) was made to be improved with the technique of hypotonic buffer combined with freeze-drying (improved group). After the acellularization process was completed, the histological structure of nerves in each group was observed by HE staining and scanning electron microscope. The interval porosity and void diameter in each group were measured by Mimics software. The biomechanical properties of nerves in each group were tested by mechanical apparatus (Endura TEC ELF3200). Results The acellularization effect of the improved chemical method with the technique of hypotonic buffer combined with freeze-drying was similar to that of the traditional Hudson method, but the histological structure was more porous in improved group than that in traditional group. The interval porosity of traditional group and improved group were 34.5% and 49.3%, respectively; the void diameter of traditional group and improved group were 11.96 and 17.61 μm, respectively. Biomechanical testing results showed that there was no statistical difference in ultimate load, ultimate stress, ultimate strain and mechanical work to fracture in each group (P>0.05). Conclusions The acellularized nerve prepared by hypotonic buffer combined with freeze-drying can be used as a new kind of nerve scaffold material to make better contribution to cell combination.