This research focused on improving the photodynamic properties of perylene through a hydrogel formulation in which PBI-DOPA cross-links chitosan chains. Photosensitive CS/PBI-DOPA hydrogel was synthesized through an oxidative cross-linking method and exhibited a biocompatible microporous structure capable of shear thinning behavior and outstanding singlet oxygen quantum efficiency. An in vitro comparative study on the phototoxic and anticancer effects of perylene tetracarboxylic dihydride (PTCDA), free PBI-DOPA and its CS/PBI-DOPA hydrogel formulation on skin cancer (A375), breast cancer (MDA- MB-231) and normal fibroblast cells were performed. The hydrogel formula showed its superiority in reducing the survival of cancer cells while minimizing toxicity in normal cells, compared to both free perylene compounds. Therefore, CS/PBI-DOPA hydrogel could be introduced as a promising biocompatible carrier system with significant efficiency for perylene delivery.