A variety of novel peptide-grafted hydrogels, of which peptides were derived from vitronectin (PQVTRGDVFTMP) or the laminin β4 chain (PMQKMRGDVFSP), were prepared in this study. The peptide-grafted hydrogels promoted the adhesion, proliferation and colony formation of hiPSCs and maintained their pluripotency up to passage 5 under xeno-free conditions. We successfully generated RPE cells from hiPSCs using one of the most suitable xeno-free peptide-grafted hydrogels, KVN2CK (KGCGGKGG-PQVTRGDVFTMP), which was derived from vitronectin, and confirmed the effect of these hiPSC-derived RPE cells in a rat retinal degeneration model (Royal College of Surgeons (RCS) rats) via subretinal transplantation, when we investigated functional improvements in vision in RCS rats after the transplantation of hiPSC-derived RPE cells. Our novel peptide-grafted hydrogels provided a safe and robust platform for generating single-layer hiPSC-derived RPE cells under xeno-free conditions, which indicates the potential of these hydrogels for stem cell therapy for retinal degenerative diseases in the future.