Extracellular Matrix (ECM) is a collective matrix surrounded by cells that allows structural and biochemical support. ECM is being extensively researched as a promising biomaterial in tissue engineering and biomedical field, due to its biocompatibility that prevents any adverse host response. Many researchers have obtained clean and effective ECM material through various decellularization processes which completely remove all DNA and cellular contents from a tissue. Although decellularized ECM has already being used successfully in clinical applications, it has not yet been applied for real use in vivo. This is because the typical decellularization process often corrupts distinct characteristics of ECM if the chemicals used are not adequately removed. In this study, supercritical fluid (SCF) extraction system was used as post-cleaning of conventional decellularization process. SC-CO2 extraction is already being used in many industrial fields, as an extraction or cleaning system. In order to remove the chemicals and the cell residues efficiently, and to preserve the distinct properties of ECM, CO2 was set into a supercritical state, over 73 atm of pressure and over 31 oC, at which point it has unique and beneficial properties of sterilization. The decellularized ECM using conventional process and post-cleaned by SCF extraction system has been evaluated and compared with the specimen without the SCF post-cleaning process. The study has shown that this novel method of using supercritical fluid extraction system has properly removed the chemicals and all cell residues from the ECM completely. Considering the superior results compared with the ECM without post-cleaning, SC-CO2 prepared ECM shows a very high potential to be actually applied for real use in vivo.