Gold Nanoparticles (AuNPs) have gained significant attention in biosensor development due to their unique physical, chemical, and optical properties. When incorporated into biosensors, AuNPs offer several advantages, including a high surface area-to-volume ratio, excellent biocompatibility, ease of functionalization, and tunable optical properties. These properties make them ideal for the detection of various biomolecules, including proteins, nucleic acids, and bacterial and viral biomarkers. Traditional methods for detecting bacteria and viruses, such as RT-PCR and ELISA, often suffer from complexities, time consumption, and labor intensiveness. Consequently, researchers are continuously exploring novel devices to address these limitations and effectively detect a diverse array of infectious pathogenic microorganisms. In light of these challenges, nanotechnology has been instrumental in refining the architecture and performance of biosensors. By leveraging advancements in nanomaterials and strategies of biosensor fabrication the sensitivity and specificity of biosensors can be enhanced, enabling more precise detection of pathogenic bacteria and viruses. This review explores the versatility of AuNPs in detecting a variety of biomolecules, including proteins, nucleic acids, and bacterial and viral biomarkers. Furthermore, it evaluates recent advancements in AuNPs-based biosensors for the detection of pathogens, utilizing techniques such as optical biosensors, lateral flow immunoassays, colorimetric immunosensors, electrochemical biosensors, and fluorescence nanobiosensors. Additionally, the study discusses the existing challenges in the field and proposes future directions to improve AuNPs-based biosensors, with a focus on enhancing sensitivity, selectivity, and their utility in clinical and diagnostic applications.