We demonstrate the first in-fiber light-induced bioactive biotin-functionalization via photobleaching fluorophore-conjugated biotin. Photobleaching the fluorophores generated free radicals that bind to the albumin-passivated inner surface of pure silica photonic crystal fiber. The subsequent attachment of dye-conjugated streptavidin to the bound biotin qualified the photo-immobilization process and demonstrated a potential for the construction of in-fiber macromolecular assemblies or multiplexes. Compared with other in-fiber bioactive coating methods, the proposed light-induced technique requires only a low-power light source, without the need for additional preactivation steps or toxic chemical reagents. This method, hence, enables a simple and compact implementation for potential biomedical applications.