Glycosaminoglycans are able to bind many growth factors and adhesive proteins, which affect cell activities such as adhesion, migration, growth and differentiation. Chondroitin sulphate, hyaluronan, sulphated hyaluronan and heparin were oxidised here (aldehyde glycosaminoglycans) to generate aldehydes on vicinal hydroxyl groups of the uronic monomers of glycosaminoglycans for subsequent direct covalent binding to amino-terminated model substrata. The properties of modified surfaces were monitored by water contact angle, zeta potential, ellipsometry measurements and atomic force microscopy showing successful immobilisation of aldehyde glycosaminoglycans. Wetting properties and zeta potentials were related to sulphate content of aldehyde glycosaminoglycans with aldehyde heparin as most wettable and negative surface and aldehyde hyaluronan as the least. The thickness of surface layers measured by ellipsometry indicated a predominant side-on immobilisation of all aldehyde glycosaminoglycans. Atomic force microscopy studies showed that immobilisation of aldehyde hyaluronan lead to a rather smooth surface coating while immobilisation of sulphated aldehyde glycosaminoglycans was characterised by a globular appearance of surfaces with higher roughness. The experiments with human fibroblast studying adhesion under serum-free conditions were carried out to learn about bioactivity of aldehyde glycosaminoglycans. It was observed that the increase in sulphation degree of aldehyde glycosaminoglycans was accompanied by increased adhesion and spreading of cells with stronger expression of focal adhesions and cytoskeletal structures. By contrast, cell adhesion and spreading were lower on aldehyde hyaluronan. Immunofluorescence staining of cells in contact with aldehyde hyaluronan revealed a stronger expression of CD44, which can represent an alternative route of cell adhesion. The results show that oxidised glycosaminoglycans can be successfully applied for the development of bioactive surface coatings. The created biomimetic microenvironment may be useful to engineer surfaces of implants and scaffolds for tissue regeneration.