Thanks to its unique biological properties, the human amniotic membrane (AM) has shown promising results for guided bone regeneration (GBR), but displays some limitations such as poor space-maintaining ability. This study thus aimed to develop a new amnion/chorion membrane (ACM), with better mechanical properties as well as comparable or improved biological properties for GBR. We first developed a new decellularization method of ACM (DL-ACM) which was validated by DNA staining and quantification, and its cytocompatibility was established in vitro. The thickness of DL-ACM was significantly increased over thirty-fivefold, and its tearing strength and compression strength significantly increased more than tenfold compared to the decellularized AM (DL-AM). In vivo, DL-ACM demonstrated its biocompatibility subcutaneously, and its osteogenic properties were compared to DL-AM and a gold standard membrane in a GBR defect model in rats. Micro-CT and histomorphometric analysis showed that DL-ACM significantly promoted early bone regeneration after 1 week and significantly increased bone regeneration compared to the empty defect and the gold standard membrane over time. In this study, we developed a simple and reproducible method to produce an acellular, non-cytotoxic, and biocompatible DL-ACM. This new membrane is as effective as AM to promote early bone regeneration while demonstrating better biomechanical properties.