This study aims to evaluate the effects of the two most widely used glycosaminoglycans (dermatan sulphate and heparin) on both the structural and biological properties of collagen-based modified membranes (COL/GAGs membranes) designed for tissue engineering. The molecular weight of dermatan sulphate and heparins was correlated with the membrane feasibility and the cell (fibroblasts and keratinocytes) ability to adhere and proliferate on the COL/GAG membranes. Microstructure and physico-chemical properties of COL/GAGs membranes were examined using scanning electron microscopy and differential scanning calorimetry; the free amino group content and the swelling properties were also detected. The morphology, proliferation and growth behaviour of keratinocytes and fibroblasts were investigated using microscopical approach and in vitro colorimetric assay. Both fibroblasts and keratinocytes are able to grow and proliferate on COL/dermatan sulphate membranes. Fibroblasts revealed significantly higher proliferation on the membranes prepared with heparin if compared to the proliferation on the membrane without heparin (COL membrane). Particularly, a combination of the membranes formulated adding high molecular weight dermatan sulphate and high molecular weight heparin could be suitable to be used as biomaterials for epidermal substitute.