At present, acellular matrix is an effective replacement material for the treatment of skin damage, but there are few systematic evaluation studies on its performance. The experimental group of this study used two decellularization methods to prepare the matrix: one was the acellular matrix which sterilized with peracetic acid first (0.2% PAA/4% ethanol solution) and then treated with hypertonic saline (group A), the other was 0.05% trypsin/EDTA decellularization after γ irradiation (group B); and the control group was soaked in PBS (Group C). Then physical properties and chemical composition of the three groups were detected. Hematoxylin eosin (HE) staining showed that the acellular effect of group B was good. The porosity of group A and B were both above 84.9%. In group A, the compressive modulus of elasticity was (9.94 ± 3.81) MPa, and the compressive modulus of elasticity was (12.59 ± 5.50) MPa in group B. There was no significant difference between group A or B and group C. The total content of collagen in acellular matrix of group A and B was significantly lower than that of group C (1. 662 ± 0.229) mg/g, but there was no significant difference in the ratio of collagen Ⅰ/Ⅲ between group B and group C. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that there was no significant difference in microstructure. Qualitative detection of fibronectin and elastin in each group was basically consistent with that in group C. Therefore, acellular matrix of group B had better performance as scaffold material. The experimental results show that the acellular matrix prepared by γ-ray sterilization and decellularization of 0.05% Trypsin enzyme/EDTA could be used for the construction of tissue-engineered skin. It could also provide reference for the preparation and mounting of heterogeneous dermal acellular matrix. It was also could be used for electrostatic spinning or three-dimensional printed tissue engineered skin scaffold which could provide physical and chemical parameters for it.