In 1959, Nobel laureate Richard Feynman suggested that ‘there is plenty of room at the bottom,’ predicting the possibility of single-molecule detection and studies. Over the last 20 years, we have witnessed a rapid development in single-molecule spectroscopy. Single-molecule spectroscopy and imaging have been demonstrated to be a powerful molecular analytical approach to studying the complex and inhomogeneous chemical, biological, and physical processes involved in protein dynamics, protein-protein interactions, protein-DNA interaction dynamics, biological and chemical catalyses, and interfacial dynamics. The discipline of single-molecule spectroscopy has been expanded across a broad range, including optical imaging, optical spectroscopy of fluorescence and Raman, atomic force spectroscopy, and various forms of scanning probe microscopy. A significant feature of this exciting development is that single-molecule spectroscopy is developing hand-in-hand with the recent advancements in nanotechnology, imaging technologies, ultrafast dynamics technologies, theoretical modeling and analyses, and computational technologies.