Expansion microscopy (ExM) is a highly effective technique for super-resolution fluorescence microscopy that enables imaging of biological samples beyond the diffraction limit with conventional fluorescence microscopes. Despite the development of several enhanced protocols, ExM has not yet demonstrated the ability to achieve the precision of nanoscopy techniques such as Single Molecule Localization Microscopy (SMLM). Here, to address this limitation, we have developed an iterative ultrastructure expansion microscopy (iU-ExM) approach that achieves SMLM-level resolution. With iU-ExM, it is now possible to visualize the molecular architecture of gold-standard samples, such as the eight-fold symmetry of nuclear pores or the molecular organization of the conoid in Apicomplexa. With its wide-ranging applications, from isolated organelles to cells and tissue, iU-ExM opens new super-resolution avenues for scientists studying biological structures and functions.