In order to overcome the disadvantages of existing treatments in heart valve tissue engineering, decellularization studies are carried out. The main purpose of decellularization is to eliminate the immunogenicity of biologically derived grafts and to obtain a scaffold that allows recellularization while preserving the natural tissue architecture. SD and SDS are detergent derivatives frequently used in decellularization studies. The aim of our study is to decellularize the pulmonary heart valves of young Merino sheep by using low-density SDS and SD detergents together, and then to perform their detailed characterization to determine whether they are suitable for clinical studies. Pulmonary heart valves of 4–6-month-old sheep were decellularized in detergent solution for 24 h. The amount of residual DNA was measured to determine the efficiency of decellularization. Then, the effect of decellularization on the ECM by histological staining was examined. In addition, the samples were visualized by SEM to determine the surface morphologies of the scaffolds. A uniaxial tensile test was performed to examine the effect of decellularization on biomechanical properties. In vitro stability of scaffolds decellularized by collagenase treatment was determined. In addition, the cytotoxic effect of scaffolds on 3T3 cells was examined by MTT assay. The results showed DNA removal of 94% and 98% from the decellularized leaflet and pulmonary wall portions after decellularization relative to the control group. No cell nuclei were found in histological staining and it was observed that the three-layer leaflet structure was preserved. As a result of the tensile test, it was determined that there was no statistically significant difference between the control and decellularized groups in the UTS and elasticity modulus, and the biomechanical properties did not change. It was also observed that decellularized sheep pulmonary heart valves had no cytotoxic effect. In conclusion, we suggest that the pulmonary valves of decellularized young Merino sheep can be used as an initial matrix in heart valve tissue engineering studies.