<p>Gold nanoparticles (AuNPs) have gained widespread use in the development of biosensors for molecular diagnostics due to their ease of functionalization and distinctive optical properties, which are attributed to plasmonic effects. Functionalization strategies are widely applied to manipulate the properties of AuNPs which are then applied for various applications including drug delivery, biosensing, and bioimaging. Among the functionalised AuNPs, nucleic acid-conjugated gold nanoparticles (AuNPs-NA conjugate) have emerged as a powerful platform for biosensing applications due to their unique optical properties, biocompatibility, and ease of functionalization. This review provides an in-depth examination of the integration of nucleic acids with AuNPs for a wide range of biosensing technologies, including detection of pathogens and biomarkers. This review also discusses various methods of conjugating nucleic acids to gold nanoparticles, including covalent bonding, electrostatic interactions, and thiol-based linkages, while highlighting the key factors that influence the stability, specificity, and sensitivity of these conjugates. The paper further explores the optical, electrochemical, and fluorescent biosensing mechanisms enabled by AuNPs-NA conjugates. Finally, it provides a perspective on the future directions of AuNPs-NA conjugates in the development of rapid, cost-effective, and highly sensitive diagnostic tools for clinical and environmental monitoring.</p>