Objective: The biological factors secreted from cells and cell-based products stimulate growth, proliferation, and migration of the cells in their microenvironment, and play vital roles in promoting wound healing. The amniotic membrane extract (AME), which is rich in growth factors (GFS), can be loaded into a cell-laden hydrogel and released to a wound site to promote the healing of the wound. The present study was conducted to optimize the concentration of the loaded AME that induces secretion of GFS and structural collagen protein from cell-laden AME-loaded collagen-based hydrogels, to promote wound healing in vitro. Materials and Methods: In this experimental study, fibroblast-laden collagen-based hydrogel loaded with different concentrations of AME (0. 1, 0. 5, 1, and 1. 5 mg/mL, as test groups) and without AME (as control group), were incubated for 7 days. The total proteins secreted by the cells from the cell-laden hydrogel loaded with different concentrations of AME were collected and the levels of GFS and type I collagen were assessed using ELISA method. Cell proliferation and scratch assay were done to evaluate the function of the construct. Results: The results of ELISA showed that the concentrations of GFS in the conditioned medium (CM) secreted from the cell-laden AME-loaded hydrogel were significantly higher than those secreted by only the fibroblast group. Interestingly, the metabolic activity of fibroblasts and the ability of the cells to migrate in scratch assay significantly increased in the CM3-treated fibroblast culture compared to other groups. The concentrations of the cells and the AME for preparation of CM3 group were 106 cell/mL and 1 mg/mL, respectively. Conclusion: We showed that 1 mg/ml of AME loaded in fibroblast-laden collagen hydrogel significantly enhanced the secretion of EGF, KGF, VEGF, HGF, and type I collagen. The CM3 secreted from the cell-laden AME-loaded hydrogel promoted proliferation and scratch area reduction in vitro.