Objective To prepare an acellular laryngo-esophagopharynx composite scaffold and explore its feasibility as an alternative material for laryngopharynx reconstruction. Methods The acellular laryngo-esophagohypopharynx composite scaffolds were prepared by perfusing detergent into the common carotid artery of adult New Zealand white rabbits and observed by HE staining, scanning electron microscope (SEM), immunofluorescence staining and biocompatibility analysis. Bone marrow mesenchyml stem cells (BMSCs) were isolated by density gradient centrifugation and seeded in the acellular scaffold. The toxicity of acellular scaffold extract was analyzed by CCK8. The scaffold with BMSCs was implanted in vivo for 1 week to observe the adhesion and proliferation of the cells in the scaffold. Results HE staining showed that there was only extracellular matrix (ECM) and no cellular components in the scaffold except cartilage. SEM revealed collagen fiber bundles of ECM, which were honeycomb without cell structure. CCK-8 assays indicated that the acellular scaffolds were not cytotoxic and BMSCs could adhere and grow in the scaffold. Conclusion It is feasible to prepare an acellular laryngo-esophagohypopharyngeal scaffold with native ECM of low immunogenicity by perfusion method.