Right ventricle outflow tract obstruction (RVOTO) is a congenital pathological condition that contributes to about 15% of congenital heart diseases. In most cases, the replacement of the right ventricle outflow in pediatric age requires subsequent pulmonary valve replacement in adulthood. The aim of this study was to investigate the extracellular matrix scaffold obtained by decellularization of the porcine pulmonary valve using a new detergent (Tergitol) instead of Triton X-100. The decellularized scaffold was evaluated for the integrity of its extracellular matrix (ECM) structure by testing for its biochemical and mechanical properties, and the cytotoxicity/cytocompatibility of decellularized tissue was assessed using bone marrow-derived mesenchymal stem cells. We concluded that Tergitol could remove the nuclear material efficiently while preserving the structural proteins of the matrix, but without an efficient removal of the alpha-gal antigenic epitope. Therefore, Tergitol can be used as an alternative detergent to replace the Triton X-100.