Objective: To prepare biofilm scaffolds using mesenteric acellular matrix and investigate their physicochemical and biological characteristics.Methods: Mesenteric cells were removed by freeze-thaw and enzymatic digestion. Mesenteries were divided into mesenteric matrix group (group A) and acellular mesenteric matrix group (group B) according to acellular or not. 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 blood vessels was detected by ultrasonography at 1m and 2m, and the blood vessels were taken for 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, and no cells remained. The expression level of DNA in group B was lower than that in group A (88.30 + 2.79ng/ml, 450.17 + 90.90ng/ml，respectively), and the difference was significant. CCK-8 cytotoxicity test showed that there was no cytotoxicity in group A and group B, FDA-PI fluorescence staining showed no cytotoxicity. Cells in group A and group B survived well, no dead cells were found; Tensile mechanics test showed that there were no significant differences in maximal force (31.41±7.09N,25.15±7.13N，respectively), maximal elongation(118.83±15.79%,111.09±15.99%，respectively), yield strength(8.35±6.46MPa,7.43±4.53MPa，respectively), yield point elongation(118.83±17.24%,95.16±22.46%，respectively) between group A and group B. The early patency of acellular mesenteric stent implantation was good, and endothelial hyperplasia was obvious in 2 months after stent implantation.Conclusion: Mesenteric cells were removed by freeze-thaw and enzymatic digestion. Mesenteric stroma was completely removed without cytotoxicity. Mesenteric stent implantation had good early patency and endothelial proliferation after 2 months.