Antibiotic delivery is important to treat bacterial infections, one of the most challenging health problems globally. This study explored the application of nucleic acids as an antibiotic-binding effector for antibiotic loading and release. The data showed that the partition coefficient of tetracycline increased proportionally to the oligonucleotide concentration ranging from 0 to 1 mM. Resultantly, the incorporation of the oligonucleotides led to enhanced tetracycline loading in the hydrogels. In addition to the enhanced drug loading, the oligonucleotides could slow the release of tetracycline from the hydrogels. Experiments were further carried out to examine the capability of oligonucleotide-functionalized hydrogels in the inhibition of bacterial growth. The results showed that the oligonucleotide-functionalized hydrogels had higher antibacterial efficiency. Moreover, after tetracycline release, the oligonucleotide-functionalized hydrogels could be refilled with fresh tetracycline to reproduce the capability of inhibiting bacterial growth. Therefore, nucleic acid oligonucleotides are a promising antibiotic-binding effector for hydrogel functionalization in antibiotic delivery.