We have shown that direct mixing of complex DNA molecular programs (scaffold + staple cocktail) in a monovalent cation-rich cell culture medium at 37 °C enables the ultra-fast, in situ self-assembly into user-defined elaborate 2D and 3D DNA nanostructures. The method was demonstrated to be particularly versatile and robust, functioning across a range of widely used biological media, including DMEM and RPMI (highly common for mammalian cell culture), E8 (for hiPSCs culture) or PBS (involved in a variety of biological protocols). Unlike conventional DNA origami assembly methods, which require hours to days, this isothermal self-assembly processus was found to be extremely rapid, well-formed DNA origamis being typically obtained in 5-15 min. As such, it can be seen as a very convenient way to prepare DNA origamis requiring nothing more than brief incubation at physiological temperature.