This report demonstrates the fi rst pH-dependent synthesis of pepsinmediated gold nanoclusters (AuNCs) with blue-, green-, and red-fl uorescent emission from Au 5 (Au 8 ), Au 13 , and Au 25 , respectively. Pepsin is a gastric aspartic proteinase (molecular weight, 34 550 g/mol) that plays an integral role in the digestive process of vertebrates. It was found that the pH of the reaction solution was critical in determining the size of Au NCs (i.e., the number of gold atoms of AuNCs). Interestingly, enzyme function of pepsin contributes to the formation of these AuNCs. The photo-stability of the Au 25 (or Au 13 ) NCs is much higher than that of Au 5 NCs (i.e., Au 25 ∼ Au 13 > > Au 5 ). The pepsin-mediated Au 25 NCs were also found to be useful as fl uorescent sensors for the detection of Pb 2 + ions by enhanced fl uorescence and the detection of Hg 2 + ions by fl uorescence quenching. Although the detailed formation mechanisms of these AuNCs require further analysis, the synthetic route using proteinase demonstrated here is promising for preparing new types of fl uorescent metal nanoclusters for application in catalysis, optics, biological labeling, and sensing.