The biotin-streptavidin bond is the strongest noncovalent bond in nature and is thus used extensively in biotechnology applications. However, the difficulty of releasing the bond without high temperatures or corrosive solutions can be a barrier to applications involving nucleic acids and other delicate substrates. Here, room-temperature phenol is employed to release biotin-tagged DNA constructs from streptavidin rapidly and efficiently. It is demonstrated that synthetic biotinylated DNA can be recovered at yields approaching 100% from both solution-phase and bead-bound streptavidin with as little as 12% (v/v) phenol, leaving the biotin tag active and reusable after extraction. As an application of this recovery method, biotinylated DNA fragments are isolated from a mixed solution to provide selectivity for solid-state nanopore detection.