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Biosynthesis of gold nanoparticles (GNPs) using fungal extracellular filtrate as the source of reducing and capping agent is reported here. The study includes the fabrication of GNPs using a soil fungus CO1 in a very effective way for material synthesis. Morphological, molecular, and phylogenetic studies of the fungus CO1 show that it is similar to the Aspergillus austwickii. Aspergillus austwickii CO1 is reported for the first time for GNPs synthesis in this paper. The characteristic surface plasmon resonance (SPR) peak for colloidal GNPs was observed at 532 nm. The GNPs remained stable for about 90 days at room temperature. The optimum temperature, pH, and substrate concentration were 100 °C, pH 9, and 1 mM AuCl4− concentration, respectively, to obtain maximum possible stability and monodispersity of synthesized GNPs. The GNPs were characterized using instrumental analysis. Transmission electron microscopy analysis confirmed the formation of spherical particles of size 14.7 ± 6.9 nm. The kinetics of the GNPs synthesis reaction was investigated by measuring particle size and zeta potential as a function of time. The stability of biosynthesized GNPs was better than chemically synthesized citrate-GNPs (Cit-GNPs) when exposed to high ionic concentration by the addition of sodium chloride in the GNP solution. The biosynthesized GNPs were able to resist aggregation even with the addition of 0.2 mM of NaCl, while Cit-GNPs aggregated at much lower concentration of 0.05 mM NaCl. Selective reactivity of the GNPs was observed toward Mg2+ ions, with a minimum detection limit of 40 ppm. Detection could be visualized by naked eye also. Thus, the non-toxic biosynthesized GNPs could further assist in the Mg2+ optical sensing application of water. [ABSTRACT FROM AUTHOR]