The cell and tissue culture substrates play a pivotal role in the regulation of cell-matrix and cellcell interactions. The surface properties of the materials control a wide variety of cell functions. Amongst various methods, layer-by-layer (LbL) assembly is a versatile surface coating technique for creating controllable bio-coatings. Here, polysaccharide/protein multilayers are proposed, which are fabricated by immersive LbL assembly and based on the chitosan /fibrinogen pair for improving the adhesion and spreading of cardiomyocytes. Two approaches in LbL assembly are employed for clarifying the effect of the bilayers order and their concentration on cardiomyocytes viability and morphology. Fourier transform infrared spectroscopy (FTIR) measurements show that the adsorption of the biopolymers is enhanced during the LbL deposition in a synergistic manner. Contact angle measurements indicate that the multilayers are alternating from less to more hydrophilic behavior depending on the biopolymer that is added last. Confocal microscopy with immunostained fibrinogen reveals that the amount of the protein is higher when the concentration of the immersion solution is increased, however, for low solution concentration it is speculated that interdigitation between the separate biopolymer layers takes place. This work motivates the use of fibrinogen in polysaccharide/protein multilayers for enhanced cytocompatibility in cardiac tissue engineering.