We report a strategy toward surface-functional polymeric microspheres using a wavelength orthogonality technique that employs photoinitiated reversible addition–fragmentation chain transfer (RAFT) dispersion polymerization and the subsequent photografting under different wavelengths of light. Initial screening of reaction conditions indicated photoreactive polymeric microsphere with uniform sizes could be prepared by using photoinitiator-functionalized macro-RAFT agents under purple light irradiation. Photoreactive polymeric microspheres allowed photografting polymerizations under UV light irradiation, and we further demonstrated the broad scope of this method by photografting acrylamide, acrylic, and methacrylic monomers. Finally, carboxyl-functionalized polymeric microspheres with an exceptional high number of carboxyl groups were successfully prepared by this technique, which permitted extensive surface bioconjugation of model proteins (e.g., streptavidin). This method should expand the capabilities of RAFT dispersion polymerization to afford diverse surface-functional polymeric microspheres for some specific applications.