G-quadruplex DNA is highly polymorphic. Its conformation transition is involved in a series of important life events. These controllable diverse structures also make G-quadruplex DNA a promising candidate as catalyst, biosensor, and DNA-based architecture. So far, G-quadruplex DNA-based applications are restricted done in aqueous media. Since many chemical reactions and devices are required to be performed under strictly anhydrous conditions, even at high temperature, it is challenging and meaningful to conduct G-quadruplex DNA in water-free medium. In this report, we systemically studied 10 representative G-quadruplexes in anhydrous room-temperature deep eutectic solvents (DESs). The results indicate that intramolecular, intermolecular, and even higher-order G-quadruplex structures can be formed in DES. Intriguingly, in DES, parallel structure becomes the G-quadruplex DNA preferred conformation. More importantly, compared to aqueous media, G-quadruplex has ultrastability in DES and, surprisingly, some G-quadruplex DNA can survive even beyond 110 degrees C. Our work would shed light on the applications of G-quadruplex DNA to chemical reactions and DNA-based devices performed in an anhydrous environment, even at high temperature.