<p>Nucleic acids (DNA and RNA) are fundamental to life, encoding biological information through linear sequences of nucleotides. Single-stranded nucleic acids, driven by complementary base pairing, fold into diverse secondary structures and subsequently form well-defined three-dimensional conformations, giving rise to specific functional sites. This leads to the emergence of functional nucleic acids (FNAs), which interact precisely with biomolecules to perform a range of biological functions beyond genetic coding, including acting as catalysts, sensors, modulators and therapeutic agents. This Review highlights the transformative impact of FNAs in advancing molecular biology and precision medicine, emphasizing their structural versatility and functional potential. We explore the production methodologies, structural principles and biological roles of key FNAs, such as aptamers and DNAzymes, along with engineering strategies aimed at optimizing their stability, affinity and catalytic efficiency. Additionally, the Review discusses the clinical trials, challenges and prospects of FNAs, positioning them as powerful molecular tools for biomedical applications that could address complex challenges in precision medicine, targeted therapies and diagnostics.</p>