Multifunctionalizable polymeric nanofibers can be tailored for various biomedical applications by selective conjugation of small molecules and bioactive ligands. This study reports the design, synthesis, and application of novel biodegradable polyester-based nanofibers bearing metal-free "clickable" handles. Polylactide-based polymers were synthesized using organo-catalyzed ring-opening polymerization to contain "clickable" chain-end functional groups that specifically react through radical or nucleophilic thiol-ene reactions. A furan-protected maleimide-containing hydroxyl-bearing initiator yielded polymers containing strained oxanorbornene unit at their chain end. In addition, postpolymerization thermal treatment provides maleimide end group-containing polymers. Solution electrospinning method was utilized to obtain bead-free nanofibers. Efficient conjugation on these nanofibers was demonstrated using metal-free conjugation reactions. It was observed that polylactide nanofibers undergo extensive biofouling, which limits their possible utilization for specific biomolecular immobilization. To alleviate this problem, polymers were modified to contain two orthogonally reactive functional groups, namely, the oxanorbornene unit and an azide group at their chain ends. The former reactive handle was used for conjugation of poly(ethylene glycol) chains to impart hydrophilicity and thus an antibiofouling ability, whereas the azide group undergoes strain-promoted azide-alkyne cycloaddition to install a protein-binding ligand such as biotin. These nanofibers were able to specifically immobilize the protein streptavidin with minimal nonspecific adsorption.