Extracellular Matrix (ECM), a natural biomaterials, have recently garnered attention in tissue engineering for their high degree of cell proliferative capacity, biocompatibility, biodegradability, and tenability in the body. Decellularization process offers a unique approach for fabricating ECM-based natural scaffold for tissue engineering application by removing intracellular contents in a tissue that could cause any adverse host responses. The effects of Supercritical carbon dioxide (Sc-CO2) treatment on the histological and biochemical properties of the decellularized extracellular matrix (de-ECM) were evaluated and compared with de-ECM from conventional decellularization process to see if it offers significantly reduced treatment times, complete decellularization, and well preserved extracellular matrix structure. The study has shown that a novel method of using supercritical fluid extraction system indeed removed all unnecessary residues and only leaving ECM. The potential of Sc-CO2 de-ECM progressed as a promising approach in tissue repair and regeneration.