As a promising approach in tissue engineering, decellularization has become one of the mostly-studied research areas in tissue engineering thanks to its potential to bring about several advantages over synthetic materials since it can provide a 3-dimensional ECM structure with matching biomechanical properties of the target tissue. Amniotic membranes are the tissues that nurture the embryos during labor. Similarly, these materials have also been proposed for tissue regeneration in several applications. The main drawback in using amniotic membranes is the limited thickness of these materials since most tissues require a 3D matrix for an enhance regeneration. In order to prevent this limitation, here we report a facile fabrication methodology for multilayered amniotic membrane-based tissue constructs. The amniotic membranes of Wistar albino rats were first decellularized with the physical and chemical methods and utilized as scaffolds. Secondly, the prepared decellularized membranes were sutured to form a multilayered 3D structure. Within the study, 7 groups including control (PBS), were prepared based on physical and chemical decellularization methods. UV exposure and freezing techniques were used as a physical decellularization methods while hypertonic medium and SDS (sodium dodecyl sulfate) protocols were used as chemical decellularization methods. The combinations of both protocols were also used. In groups, A was the control and group B was applied just UV. In group C was applied UV and freezing. In addition to UV and freezing, in group D was applied hypertonic solution while group E was applied SDS (0.03 %). In group F was applied UV, freezing, hypertonic solution and SDS (0.03 %). In group G was applied UV, hypertonic solution, SDS (0.03 %) and freezing, respectively. Based on the histological and quantitative analyses, F and G groups were found as the most efficient decellularization protocols in rat amniotic membranes. Then, group F and G decellularized amniotic membranes were used to form scaffolds and thus-formed matrices were further characterized in vitro cell culture studies and mechanical tests. Cytotoxicity analyses performed using MTT showed a good cell viability in F and G groups scaffolds. The percentage viability rate was higher in G group (81.3 %) compared to F (75.33 %) and also cell viability in G group was found more meaningful according to p value which was obtained 0.007. Cellular adhesions after in vitro cell culture and morphology of scaffolds were evaluated by scanning electron microscopy (SEM). It was observed that the cells cultivated in equal amounts of tissue scaffolds were higher in the F compared to that observed in group G. The mechanical testing with 40 N force revealed 0.77 mm displacement in group F while it was 0.75 mm in group G. Moreover, according to forcecontrolled test, 2.9 mm displacement of F group and 1.2 mm displacement of G group was measured. As a result, this study shows that the multilayered decellularized amniotic membrane scaffolds support cell survival and adhesion and can form a flexible biomaterial with desired handling properties.