In the past few decades, biosensors have been gradually developed for the rapid detection and monitoring of human diseases. Recently, functional nucleic-acid (FNA) biosensors have attracted the attention of scholars due to a series of advantages such as high stability and strong specificity, as well as the significant progress they have made in terms of biomedical applications. However, there are few reports that systematically and comprehensively summarize its working principles, classification and application. In this review, we primarily introduce functional modes of biosensors that combine functional nucleic acids with different signal output modes. In addition, the mechanisms of action of several media of the FNA biosensor are introduced. Finally, the practical application and existing problems of FNA sensors are discussed, and the future development directions and application prospects of functional nucleic acid sensors are prospected.