Advancement of super-resolution fluorescence microscopy (SRM) has recently allowed applications to the biosensing by offering significant advantages over conventional methods. Its nanoscale spatial resolution and single-molecule sensitivity allow visualization and quantification of biomolecular targets without the need of signal amplification steps typically required in traditional biosensing methods. Moreover, recent innovations in probe design and imaging protocols have expanded SRM capabilities to enable dynamic biosensing in living cells, revealing molecular processes in their native cellular contexts. In this review, we discuss these applications of various SRM techniques to biosensing by highlighting their unique capabilities in providing spatial distribution information and high molecular sensitivity. We address several challenges that must be overcome for the broader application of SRM-based biosensing. Finally, we discuss perspectives on future directions for advancing this field towards practical applications.