Genetically site-specific introduction of noncanonical amino acids (ncAAs) for protein conjugation generally requires incorporation through exogenous feeding of chemically synthesized ncAAs. We developed a p-amino-phenylalanine (pAF)-based relay system that enables site-specific functionalization of proteins without chemical synthesis of the building blocks. pAF was biosynthesized under optimized conditions, followed by site-specific incorporation into a specific protein residue. The resulting protein was ready for functionalization using an oxidative conjugation reaction. We demonstrated the use of this relay system by preparing a fluorophore-labeled anti-HER2 single-chain variable fragment antibody for fluorescent imaging.