Technologies based on digital microfluidics (DMF) have made significant advancements in the automated manipulation of microscale liquids and complex multistep processes. Due to their numerous benefits, such as automation, speed, cost-effectiveness, and minimal sample volume requirements, these systems are particularly well suited for immunoassays. In this review, an overview is provided of diverse DMF manipulation platforms and their applications in immunological analysis. Initially, droplet-driven DMF platforms based on electrowetting on dielectric (EWOD), magnetic manipulation, surface acoustic wave (SAW), and other related technologies are briefly introduced. The preparation of DMF is then described, including material selection, fabrication techniques and droplet generation. Subsequently, a comprehensive account of advancements in the integration of DMF with various immunoassay techniques is offered, encompassing colorimetric, direct chemiluminescence, enzymatic chemiluminescence, electrosensory, and other immunoassays. Ultimately, the potential challenges and future perspectives in this burgeoning field are delved into.