Nucleic acids, which are fundamental to living organisms, play crucial roles in carrying and transmitting genetic information. Advances in molecular biology have led to the exploration of functional nucleic acids (FNAs), including aptamers, DNAzymes, and G-quadruplexes, which are known for specific recognition or catalysis. FNAs, with high specificity, sequence programmability, modification ease, and biocompatibility, have extensive applications in biosensing, environmental monitoring, drug delivery, and cancer diagnosis. This review focuses on the structure and specific recognition principles of FNAs from the perspective of specific recognition, followed by an exploration of biosensing and biomedical applications, offering insights into current challenges and future trends in FNAs as recognition elements.