Gene therapy, siRNA, and therapeutic aptamers attract great interest owing to their versatility to treat a wide range of diseases and their potential high selectivity. Unfortunately, oligonucleotide-based therapeutics suffer rapid degradation by nucleases, scarce cell internalization, and fast kidney clearance. To address these limitations, the covalent attachment by mild chemical reactions of an activated polyethylene glycol (PEG) is widely used to obtain PEGylated nucleic acids showing a more favorable pharmacokinetic profile. We describe here a method for the enzymatic formation of PEGylated nucleic acids employing T4 DNA ligase: the ligation protocol was set up and optimized allowing the complete achievement of PEGylated oligonucleotides amenable to further enzymatic reactions. The feasibility of this approach for bioconjugation was demonstrated employing a set of PEG-donors and oligonucleotide acceptors, differing in the chemical link between PEG and the oligonucleotide donor, and in the length, sequence, and structure of the oligonucleotides employed. The ligase reaction allowed us to obtain double-stranded as well as single-stranded oligonucleotides, thus demonstrating the applicability of the method to a variety of substrates suitable for diagnostic and therapeutic applications.