Burn injury has been reported to be an important cause of morbidity and mortality and it is still considered as unmet clinical need. Although there is a myriad of effective stem cells suggested for skin regeneration, there is no one ideal scaffold. The aim of this study was to develop a 3D bi-layer scaffold made of biological decellularized human amniotic membrane (AM) with viscoelastic electrospun nanofibrous silk fibroin (ESF) spun on top. The fabricated 3D bi-layer AM/ESF scaffold was submerged in ethanol to induce β-sheet transformation as well as to get a tightly coated and inseparable bilayer. The biomechanical and biological properties of the 3D bi-layer AM/ESF scaffold were investigated. The results indicate a significant improved mechanical properties of the AM/ESF compared to the AM alone. Both AM and AM/ESF possess a variety of suitable adhesion cells without detectable cytotoxicity against the Adipose Tissue-Derived Mesenchymal Stem Cells (AT-MSCs). The AT-MSCs show increased expression of two main pro-angiogenesis factors VEGFa and bFGF when cultured on the AM/ESF for 7 days in comparison with AM alone. The results suggest that AM/ESF scaffold with autologous AT-MSCs has excellent cell adhesion and proliferation along with production of growth factors which serves as a possible application in clinical setting in skin regeneration.