Highly specific and tight-binding nucleic acid aptamers have been selected against a variety of molecular targets for over 20 years. A significant proportion of these oligonucleotides display G-quadruplex structures, particularly for DNA aptamers, that enable molecular recognition of their ligands. G-quadruplex structures couple a common scaffold to varying loop motifs that act in target recognition. Here, we review DNA G-quadruplex aptamers and their ligands from a structural and functional perspective. We compare the diversity of DNA G-quadruplex aptamers selected against multiple ligand targets, and consider structure with a particular focus on dissecting the thrombin binding aptamer – thrombin interaction. Therapeutic and analytical applications of DNA G-quadruplex aptamers are also discussed. Understanding DNA G-quadruplex aptamers carries implications not only for therapeutics and diagnostics, but also in the natural biochemistry of guanine-rich nucleic acids.