Basement membrane (BM) has been investigated widely and applied in the field of bio-synthesized scaffold design in tissue engineering. However, this respect of investigation has seen scarcely in the field of esophageal tissue engineering. This study reports BM's basic topographic characters and quantification of structural and major proteins involving collagen IV, laminin, entactin, proteoglycans of porcine esophagus. Several methods were adopted to strip epithelium from mucosa tissue. For example, ethylene diamine tetraacetic acid, sonication, dithiothreitol, cold sodium chloride (NaCl), mechanical force of glass coverslip, and cold trypsin and so forth. Assessed experimental results under different conditions, the optimal condition to isolate epithelium from BM was established. After the reaction of cold trypsin at 4°C for 15 h, the BM was isolated from epithelium and exposed integratedly. Hematoxylin and eosin staining and immunohistochemical staining verified the effectiveness of epithelium removal and the integrity of BM. The topographic features of the exposed BM were observed under transmission electron microscopy and scanning electron microscopy. It displayed a rugged surface and 3-D topography consisting of pores and fibers with sub-100 nm range via ImageJ software. The major proteins existing in BM were quantitatively analyzed by enzyme-linked immune sorbent assay. All these results will provide references for the design of synthesized scaffolds and protein modification in esophageal tissue-engineering research. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.