In the last three decades, the fast development of single-molecule techniques has revolutionized the way we observe and understand biological processes. Some of these techniques have been further adapted as tools for bioanalysis. This book summarizes and details the frontiers of the development of these tools as well as their applications. The contributors are young and established researchers in their respective fields. The main content originates from the lecture notes of a chemistry graduate course taught by the book editor at Nanjing University. This book is suitable to be used as a textbook for a high-level undergraduate or an entry-level graduate course. The systematically written content provides a thorough illustration of the mechanisms of each methodology presented.