Nucleic acids, which are fundamental to living organisms, play a crucial role 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, 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, followed by an exploration for biosensing and biomedical applications, offering insights into current challenges and future trends in FNAs as recognition elements.