Objective To prepare a pancreatic decellularized bio-derived scaffold(PDBC) in vitro, providing a novel natural bio-derived scaffold for islet and pancreatic tissue engineering. Methods The pancreas from adult SD rats was obained. To prepare the PDBC, we used physical freeze thawing, socking and perfusing that based on retrograde pancreatic and bile ducts’ perfusion. After decellularization, normal pancreatic tissue and PDBC were examined by genomic DNA quantification, and through HE staining,transmission electron microscope and immunohistochemistry.The conservation of collagen type Ⅳ, fibronectin and laminin were identified. Results BR>The DNA gelelectrophoresis showed that no bands in the experimental group while a 100bp band appeared in the control group. Compared with the DNA in the control group, BR>the DNA in PDBC was less than 5%. Histochemical staining and transmission electron microscopy indicated that PDBC preserved the complete extracellular matrix and no cells attached to PDBC surface.Immunofluorescence results demonstrated that collagen, elastin and other extracellular components were well preserved, without obvious nuclear components. Conclusion The PDBC prepared by freeze thawing, socking and perfusing was decellularized completely and preserved the ext