Controlling the adhesion of mammalian cells at the interface between materials and biological environments is a real challenge when designing materials for tissue engineering applications. The surface properties of implanted materials are known to have a significant impact on cell adhesion. Herein, two types of polyelectrolyte multilayers (PEMs) hydrogel membrane based on marine-derived polysaccharides of chitosan (CHI) and alginate (ALG) biopolymers were fabricated by the layer-by-layer (LbL) technique using simple approach involving the change in assemble sequence of chitosan with different degree of deacetylation (DD). The effect of PEMs formation on the surface properties and their effects on the adhesion of mouse fibroblast cell (L929) of the two membranes were studied. The results showed that the formations of ALG/CHI membranes were related to the rigidity and conformations of chitosan molecules. The adhesion of L929 cell was strongly depended on the surface roughness rather than stiffness. The surface of PEMs can be strongly cytophilic (cell adhesive, terminated with chitosan (C)) or strongly cytophobic (cell resistant, terminated with chitosan (C)). The results obtained indicate that ALG/CHI PEMs with different surface morphology and roughness could be used in vitro to manipulate cell behaviors to improve upon the design of tissue-engineered membranes.