Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.