This report demonstrates the first pH-dependent synthesis of pepsin-mediated gold nanoclusters (AuNCs) with blue-, green-, and red-fluorescent 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 similar to Au-13 > > Au-5). The pepsin-mediated Au-25 NCs were also found to be useful as fluorescent sensors for the detection of Pb2+ ions by enhanced fluorescence and the detection of Hg2+ ions by fluorescence 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 fluorescent metal nanoclusters for application in catalysis, optics, biological labeling, and sensing.