In recent years, digital microfluidics (DMF) has emerged as a powerful tool for the manipulation over individual droplets in integrated microfluidic devices. In DMF, discrete droplets at low volume can be individually controlled to merge, mix, split, and dispense from reservoirs through an array of electrodes. Apart from possessing the advantages of conventional microfluidics, including minimized reagent use and reaction time, prevention of cross-contamination as well as a high level of integration, DMF is also capable of maintaining precise control over unit droplets without the use of propulsion devices such as pumps or mechanical mixers, which cannot be made possible in traditional channel-based microfluidics. The flexibility of DMF also makes it an ideal platform for integration with biosensors, which allows for in-line detection in a fully integrated system. These unique properties have allowed DMF to be used for multiplexed detection of biological and chemical reactions at microscale or nanoscale. In fact, it has been exploited in a large variety of application scenarios, including cell-based applications, DNA-based applications, and protein-based applications. DMF devices have also opened many new possibilities in point-of-care diagnostics. In this chapter, we first introduce the basic theory of DMF devices and analyze the mechanism of droplet actuation. Two common configurations (parallel plate and single plate) of DMF devices as well as their fabrication methods have also been described. Furthermore, a detailed discussion focusing on the integration of biosensors into the DMF systems is given for biological applications, including both electrochemical biosensors and optical biosensors. Various application scenarios based on these devices have also been summarized. Finally, we summarize the advantages of DMF devices and challenges facing their development in the future.