Objective To optimize human acellular dermal matrix(ADM) and evaluate its biological characters. Methods Human skin was treated with hypertonic saline followed by NaOH maceration(group A), hypertonic saline followed by sodium dodecyl sulfate (SDS) detergent(group B) or Dispase Ⅱ followed by Triton X-100(group C), the resulting ADM were sectioned, and then were stained by special immunohistochemistry method. The cytotoxicity of them were evaluated by methyl thiazolyl tetrazolium (MTT) colorimetry and then cell compatibility was analyzed by cell culture;The optimized ADM resulted was choosen for use. Fibrablasts(FBs)were transfected with adenovirus vector encoding green fluorescent protein gene(Ad-GFP)and the growth of them on the optimized ADM was observed by fluorescent microscopy. Results Collagen and elastic fibers can still be observed in three kinds of ADM. The cells in dermis can be disintegrated both in group A and C, but not in group B. The cytotoxicity scores of the ADM prepared in group A and B were grade 0 or grade 1, while that of group C was more than grade 1.The ADM prepared by NaCl-NaOH maceration had good biocompatibility. There was statistical difference in adhering number of NIH3T3 cells in group A and B. NIH3T3 cells grew well in group A and the resulted ADM was optimized. FBs transfected with Ad-GFP grew well in the optimized ADM. Conclusion The ADM prepared by NaCl-NaOH maceration was a good tissue engineering biomaterial with a little cytotoxicity and rich in resouce.