As the technology advances, a growing number of biomedical microelectromechanical systems (bio-MEMS) research involves development of lab-on-a-chip devices and micrototal analysis systems. For example, a portable instrument capable of biomedical analyses (e.g., blood sample analysis) and immediate recording, whether the patients are in the hospital or home, would be a considerable benefit to human health with an excellent commercial viability. Digital microfluidic (DMF) system based on the electrowetting-on-dielectric (EWOD) mechanism is an especially promising candidate for such point-of-care systems. The EWOD-based DMF system processes droplets in a thin space or on an open surface, unlike the usual microfluidic systems that process liquids by pumping them in microchannels. Droplets can be generated and manipulated on EWOD chip only with electric signals without the use of pumps or valves, simplifying the chip fabrication and the system construction. Microfluidic operations by EWOD actuation feature precise droplet actuation, less contamination risk, reduced reagents volume, better reagents mixing efficiency, shorter reaction time, and flexibility for integration with other elements. In addition, the simplicity and portability make the EWOD-based DMF system widely popular in biomedical or chemical fields as a powerful sample preparation platform. Many chemical and biomedical researches, such as DNA assays, proteomics, cell assays, and immunoassays, have been reported using the technology. In this paper, we have reviewed the recent developments and studies of EWOD-based DMF systems for biomedical applications published mostly during the last 5 years.