Abstract: Objective To explore the feasibility of preparing ureteral acellular matrix (UAM) using perfusion systems. Methods Using the luminal structure of the ureter, the UAM was prepared by perfusing canine ureter with SDS, TritonX-100, or both. The residual nuclei in the UAM were evaluated using HE staining, DAPI staining, DNA quantification, and agarose gel electrophoresis. The three-dimensional ultrastructure and the bioactive components were evaluated by Masson’s trichrome staining, Alcian Blue staining, collagen quantification, GAG quantification, scanning electron microscopy (SEM), and toxicity detection. Results HE staining and DAPI staining showed the absence of obvious nuclear materials in the combined group, which was further confirmed by DNA quantification and agarose gel electrophoresis. Masson’s trichrome staining, Alcian Blue staining, collagen quantification and GAG quantification all verified that the ultrastructure and the bioactive components were well preserved in the combined group. SEM showed a large amount of porous structure on the surface of the UAM prepared by combined perfusion, and toxicity assay confirmed that the prepared UAM was nontoxic. Conclusion Perfusion of canine ureter with SDS and TritonX-100 is feasible to prepare UAM for ureteral reconstruction.