Extracellular matrix (ECM) plays pivotal roles in developments, wound healing, migration, angiogenesis, and other tissue developments. Developing new ECM may give us an innovative approach of controlling the cell and tissue behavior, regenerating damaged tissues, and other biotechnology usages. Here we report new ECM like biomaterials, pFZC, obtained from the fertilized Zebrafish chorion (FZC). pFZC was prepared by digesting the fertilized Zebrafish chorion with pronase. Differentiation of P19 embryonic carcinoma cells (P19 EC) on pFZC demonstrated that the level of the differentiation of P19 EC was comparable to cells on gelatin or fibronectin coated cover slide. Analyses of glycosylates of Zebrafish chorions using various Lectins revealed that during the fertilization, structurally organized glycosylations of Zebrafish chorion were increased and FZC was glycosylated with mannose, fucose, n-acetylglucosamine, and sialic acid. In addition, after FZC proteolytic digestion with pronase, the digested FZC proteins, pFZC, displayed similar glycosylation manner. SDS gel electrophoresis and mass spectroscopy analysis of pFZC revealed that the major component of pFZC is ZP2 protein. Newly identified ECM like biomaterials, pFZC, will provide more broad ways of controlling the cell and tissue environments.