Objective To prepare biofilm scaffolds by using mesenteric acellular matrix, so as to investigate their physicochemical and biological characteristics. Methods The mesenteric tissues were subjected to trypsin digestion, and the mesenteric cells were removed after repeated freezing and thawing of mesenteric tissue. Mesenteries were divided into mesenteric matrix group (Group A) and acellular mesenteric matrix group (Group B). The physical and chemical properties of mesenteric matrix in two groups were tested by HE staining, electron microscopy, DNA detection, cytotoxicity test and tensile mechanics test. The blood flow of the vessels was detected by ultrasonography at 1st week, 1st month and 2nd month, and the vessels were observed by pathological examination. Results HE staining and electron microscopy showed that the mesentery of Group B was loose in acellular mesentery matrix, and the arrangement of fibers was neat, with no cells remaining. Compared with Group A, the expression level of DNA in Group B was lower, with more completely decellularized cells. CCK-8 cytotoxicity test showed that there was no cytotoxicity in Group A and Group B. FDA-PI fluorescence staining showed no cytotoxicity of cells in both groups. Cells in Croup A and Group B survived well, and no dead cells were found. Tensile mechanics test showed that there were no significant differences in maximum tensile force, maximum elongation, yield strength, yield point elongation between Group A and Group B. The early patency of acellular mesenteric stent implantation was good, and endothelial hyperplasia was obvious at 2nd month after stent implantation. Conclusion sMesenteric cells were removed by freeze-thaw and enzymatic digestion. Mesenteric stroma was completely removed without cytotoxicity, which showed good mechanical characteristics. Mesenteric stent implantation had good early patency and endothelial proliferation after 2 months.