Cell membranes are complicated multicomponent structures, related to many basic cellular processes, such as substance transporting, energy conversion, signal transduction, mechanosensing, cell adhesion and so on. However, cell membranes have long been difficult to study at a single-molecule level due to their complex and dynamic properties. During the last decades, biophysical imaging techniques, such as atomic force microscopy and super-resolution fluorescent microscopy, have been developed to study biological structures with unprecedented resolution, enabling researchers to analyze the composition and distribution of membrane proteins and monitor their specific functions at single cell/molecule level. In this review, we highlight the structure and functions of cell membranes based on up-to-date biophysical techniques. Additionally, we describe the recent advances in force-based detecting technology, which allow insight into dynamic events and quantitativelymonitoring kinetic parameters for trans-membrane transporting in living cells.