Applications of microelectromechanical systems (MEMS) technology are widespread in both industrial and research fields providing miniaturized smart tools. In this review, we focus on MEMS applications aiming at manipulations and characterization of biomaterials at the single molecule level. Four topics are discussed in detail to show the advantages and impact of MEMS tools for biomolecular manipulations. They include the microthermodevice for rapid temperature alternation in real-time microscopic observation, a microchannel with microelectrodes for isolating and immobilizing a DNA molecule, and microtweezers to manipulate a bundle of DNA molecules directly for analyzing its conductivity. The feasibilities of each device have been shown by conducting specific biological experiments. Therefore, the development of MEMS devices for single molecule analysis holds promise to overcome the disadvantages of the conventional technique for biological experiments and acts as a powerful strategy in molecular biology.