Since the disclosure of DNA/RNA as the genetic material, nucleic acid analysis has been an indispensable category in molecular diagnosis, disease prediction, prognosis, and fundamental biological research. Among various platforms for the analysis of DNA/RNA, electrowetting-based digital microfluidics (DMF) has emerged as a promising tool. Using electrical signals to manipulate individual droplets, DMF devices could be packaged into compact automatic systems for multiplex analytical assays in a miniaturized format, especially suitable for point-of-care applications. Herein, we review basic technical principles that support nucleic acid analysis on DMF platforms and recent advances in applications regarding nucleic acid extraction, amplification, and further analysis such as sequencing on DMF. We also discuss the commercialization status of related technology and applications, alongside our opinions on the future development of digital microfluidics for nucleic acid analysis.