Functionalized oligonucleotides (ONs) are widely applied as target recognition molecules for biosensing and gene regulation. Herein, we describe a general method for post-synthetic modification of ONs based on the oxidative amination of 4-thio-2'-deoxyuridine (4SdU) with sodium periodate and several amines. Alkyne-/azide-, biotin-, and fluorophore-modified ONs were prepared by modifying 4SdU-containing ONs with the corresponding amines and characterized for their bioorthogonal reactivity, streptavidin-binding affinity, and fluorescence properties, respectively. We synthesized three fluorophore-modified ONs with and without the aromatic fluorophores conjugated to modified nucleobases and investigated their emission properties. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Post-synthetic modification of ONs Supporting Protocol: Preparation of dansyl ethylenediamine Basic Protocol 2: Quantification of ON reaction yields Basic Protocol 3: Characterization of modified ONs.