Antifouling polymer layers containing extracellular matrix-derived peptide motifs offer promising new options for biomimetic surface engineering. In this contribution, we report the design of antifouling vascular grafts bearing biofunctional peptide motifs for tissue regeneration applications based on hierarchical polymer brushes. Hierarchical diblock poly(methyl ether oligo(ethylene glycol) methacrylate-block-glycidyl methacrylate) brushes bearing azide groups (poly(MeOEGMA-block-GMA-N3)) were grown by surface-initiated atom transfer radical polymerization (SI-ATRP) and functionalized with biomimetic RGD peptide sequences. Varying the conditions of copper-catalyzed alkyne-azide “click” reaction allowed for the immobilization of RGD peptides in a wide surface concentration range. The synthesized hierarchical polymer brushes bearing peptide motifs were characterized in detail using various surface sensitive physicochemical methods. The hierarchical brushes presenting the RGD sequences provided excellent cell adhesion properties and at the same time remained resistant to fouling from blood plasma. The synthesis of anti-fouling hierarchical brushes bearing 1.2 × 103 nmol/cm2 RGD biomimetic sequences has been adapted for the surface modification of commercially available grafts of woven polyethylene terephthalate (PET) fibers. The fiber mesh was endowed with polymerization initiator groups via aminolysis and acylation reactions optimized for the material. The obtained bioactive antifouling vascular grafts promoted the specific adhesion and growth of endothelial cells, thus providing a potential avenue for endothelialization of artificial conduits.