Fluorescence super-resolution microscopy has undergone several rounds of innovation and development since the 1990s. The spatial resolution of fluorescence super-resolution microscopy exceeds the diffraction limit by a wide margin, achieving a lateral resolution below to ~20 nm and enabling imaging and tracking at the molecular level. The latest generation of ultrahigh-resolution microscopy is the product of extensive development and innovative combination of conventional super-resolution techniques. This review aimed to introduce the new generation of fluorescence super-resolution microscopy at a sub-20-nm scale lateral resolution, and we also elucidated their similarities and differences compared with the conventional super-resolution techniques. Additionally, this review explored how emerging technologies optimize optical systems, scanning strategies, and sample preparation to improve resolution. Finally, we discussed the potential applications and development of high-resolution fluorescence microscopy in biomedical research.