Recent advances in nanotechnology and optical imaging have transformed molecular diagnostics, enabling the detection and analysis of individual biomolecules with unprecedented precision. Nanobiosensors provide ultrasensitive molecular detection, and super-resolution microscopy (SRM) exceeds the diffraction limit of conventional optics to achieve nanometer-scale resolution. Although their integration remains in its infancy, with only a handful of proof-of-concept studies reported, the convergence of nanobiosensors and SRM holds significant promise for next-generation diagnostics. In this review, we first outline nanobiosensor-based single-molecule detection strategies and highlight representative implementations. These include plasmonic–SRM hybrids, electrochemical–optical correlatives, and SRM-enabled immunoassays, with a focus on their applications in oncology, infectious diseases, and neurodegenerative disorders. Then, we discuss emerging studies at the interface of nanobiosensors and SRM, including nanostructure-assisted SRM. Despite not being true biosensing approaches, these studies provide valuable insights into how engineered nanomaterials can improve imaging performance. Finally, we evaluate current challenges, including reproducibility, multiplexing, and clinical translation, and outline future opportunities, such as the development of photostable probes, artificial intelligence-assisted image reconstruction, microfluidic integration, and regulatory strategies. This review highlights the synergistic potential of nanobiosensors and SRM, outlining a roadmap toward clinically translatable next-generation single-molecule diagnostic platforms.

Recent advances in nanotechnology and optical imaging have transformed molecular diagnostics, enabling the detection and analysis of individual biomolecules with unprecedented precision. Nanobiosensors provide ultrasensitive molecular detection, and superresolution microscopy (SRM) exceeds the diffraction limit of conventional optics to achieve nanometer-scale resolution. Although their integration remains in its infancy, with only a handful of proof-of-concept studies reported, the convergence of nanobiosensors and SRM holds significant promise for next-generation diagnostics. In this review, we first outline nanobiosensor-based single-molecule detection strategies and highlight representative implementations. These include plasmonic–SRM hybrids, electrochemical–optical correlatives, and SRM-enabled immunoassays, with a focus on their applications in oncology, infectious diseases, and neurodegenerative disorders. Then, we discuss emerging studies at the interface of nanobiosensors and SRM, including nanostructure-assisted SRM. Despite not being true biosensing approaches, these studies provide valuable insights into how engineered nanomaterials can improve imaging performance. Finally, we evaluate current challenges, including reproducibility, multiplexing, and clinical translation, and outline future opportunities, such as the development of photostable probes, artificial intelligence-assisted image reconstruction, microfluidic integration, and regulatory strategies. This review highlights the synergistic potential of nanobiosensors and SRM, outlining a roadmap to clinically translatable, next-generation single-molecule diagnostic platforms.