Fluorescence microscopy technology is a crucial tool in biomedical research, enabling us to visualize the structure and function of tissue cells at the cellular level. Its great potential lies in fluorescence super-resolution fluorescence microscopy, which surpasses the limitations of diffraction and enables high-resolution real-time imaging of nano-subcellular structures, including organelles and cell matrices. It, therefore, contributes significantly to exploring disease mechanisms, ranging from the exchange of protein aggregates at a structural level to the identification of morphological defects in organelles. In this review, we first provide an overview of the principles of various super-resolution microscopy techniques. We then delve into the intricate transmembrane interactions exhibited by membrane organelles, as well as the intricate information communication within membraneless organelles in both physiological and pathological conditions. Finally, we examine the evolution of super-resolution technology and discuss its application in biomedicine. Overall, this review briefly introduces the principles of super-resolution microscopy and highlights its novel results in organelles to guide its application in the biomedical field.