<p>In this study, isocitrate dehydrogenase (IDH) was employed exclusively as a structural protein template for the synthesis of gold nanoparticles, rather than relying on its enzymatic catalytic function. The reduction of Au³⁺ to Au⁰ is accomplished through the intrinsic reducing capability of amino acid residues (particularly tryptophan and tyrosine) within the protein scaffold, which simultaneously acts as a capping and stabilizing agent. This protein-templating strategy provides a straightforward, biocompatible, and cofactor-free approach for developing AuNP-based biosensing platforms. So, the IDH-based AuNPs offers promising applications across medical diagnostics, environmental monitoring, food safety, and research. In this study, we produced the recombinant isocitrate dehydrogenase of <i>Yarrowia lipolytica</i> yeast in <i>E. coli BL21.</i> After optimizing the enzyme activity/stability in the proper pH and temperature, its activity was then measured with K_m of 50.1 µM and 9.7 µM for isocitrate and NADP⁺, respectively. Then, we synthesized gold nanoparticles with a size of 16.3&#xa0;nm and investigated their synthesis in the absence and presence of IDH. The in situ synthesized AuNPs showed acceptable results in the presence of IDH during the emergence of ruby red color or the detection of increased Au⁰ at 526&#xa0;nm. IDH facilitates in situ AuNP formation through a combination of: (i) enzymatic generation of NADPH, (ii) reducing amino acid residues on the protein surface, and (iii) a templating effect that controls nucleation. This multifunctional protein matrix eliminates the need for post-synthesis enzyme attachment, offering a streamlined platform for biosensor development.</p> Graphical Abstract <p></p>