Strategies to generate platforms combining tissue targeting and regeneration properties are in great demand in the regenerative medicine field. Here we employ an approach to directly visualize the immobilization of cysteine-terminated peptides on a novel fluorogenic surface. Peptides with relevant biological properties, CLPLGNSH and CLRGRYW, were synthesized to function as peptide binders to transforming growth factor (TGF)-β1 and collagen type II (CII). The selective immobilization of the peptides was directly detected using a fluorogenic surface. Adhered proteins were confined to patterns of these peptides matching with the fluorogenic areas. These results show that the fluorogenic signal can be used to detect the chemo-selective immobilization of non-fluorescent biomolecules and to correlate the cell response with the patterned peptides. After analyzing the sequence specificity and cross-reactivity of the binding of TGF-β1 and CII to the respective peptide regions employing immunofluorescence assays, both peptides were co-immobilized in a step-wise process as detected by the fluorogenic surface. TGF-β1 and CII could be self-sorted from a mixture in a regio-selective manner resulting in a bi-functional protein platform. Surfaces of CLPLGNSH pre-loaded with TGF-β1 showed excellent bioactivity in combination with human articular chondrocytes (HACs) and stimulated expression of chondrogenic markers.