Fluorescent insulin fibrils gold nanoclusters (Au NCs) have been synthesized through the reduction of gold by human insulin in fibrillated form. Likewise, nanocluster formation has been regulated by insulin, working as a protein-based template. Environment- and surface-controlled experiments have shown the optimized synthesis conditions is comprised of a pure aqueous alkaline solvent for insulin under constant heat at physiological temperature (37 °C) prior to addition of the Au precursor (HAuCl4), followed by subsequent heating (37 °C) and vigorous stirring after the addition of HAuCl4 until the completion of the synthetic approach. Microscopy experiments detected the presence of primordial fibril structures in samples of heated human insulin in the alkaline medium prior to addition of HAuCl4, while encountering more developed insulin fibrils in the terminal production of Au NCs. This investigation provides insight to the development of a novel synthesis of Au NCs in the alkaline medium, while providing a graphical description of the environmental and surface-dependent effects that were presented in the synthesis of human insulin nanoclusters. The study provides pertinent information for future synthetic procedures, as the protein state of several protein-nanoparticle systems may reflect on the results that were obtained herein.