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The unique tunable physicochemical properties of gold nanoparticles (AuNPs). In addition to their excellent biological compatibility, conducting properties, and high surface-to-volume ratio make them ideal candidates for electronic signal transduction of biological recognition events in biosensing platforms. Commonly, AuNPs are synthesized by chemical reduction of HAuCl4 and the introduction of a protective agent (stabilizer). In recent years, alternative biosynthetic approaches have been explored using microorganisms as bionanofactories to produce metal nanoparticles. AuNP biosynthesis procedures include the use of fungi and bacteria strains. In this study, the alkalothermophilic actinomycetes Thermomonospora curvata, Thermomonospora fusca, and Thermomonospora chromogena were used for the extracellular biosynthesis of AuNP. Optimal growth and biosynthesis conditions were established for each microorganism. The average AuNP size obtained was in the range of 30-60 nm. The AuNP were characterized using UV-Vis spectra, transmission electron microscopy images, and particle-size distribution. The obtained particles were monodisperse and water soluble. In order to improve stability, glutaraldehyde was used to functionalize the AuNP after synthesis. The green-chemistry AuNP obtained in this study can potentially be used to enhance biosensing applications: as transducers or electroactive labels, especially in nanoparticle-based electrochemical DNA detection systems.