Single molecule detection is a technology of studying biomolecules with high spatial and temporal resolution. By exploiting recent technical advances, we are able to observe, detect, even manipulate individual molecules and study their conformational changes and dynamic behaviors. New information can be obtained from the single molecule research, which is otherwise hidden or averaged out. In recent years, the development of single molecule detection techniques has opened up a new era of life science. In this review, we introduce the advances of the techniques for detecting single molecules in cell biology and review the development of single molecule detection in living cells.