Owing to diverse functionalities and metal binding abilities, proteins have been proven to be promising ligands in the synthesis of gold nanoclusters (Au NCs). In this work, we explored β-lactoglobulin (β-Lg), a protein byproduct generated during cheese processing, as a biotemplate for fabrication of Au NCs by a facile and green method for the first time. The as-prepared Au NCs are water soluble and highly fluorescent and exhibit high sensitivity and selectivity for Hg2+ detection in aqueous solution. Interestingly, we found that the fluorescence of these Au NCs is stable either in a variety of complex matrixes or over a broad pH range (5.0-13.0) and therefore can be explored as a cell and animal imaging agent. More importantly, we demonstrated that the β-lactoglobulin-stabilized Au NCs (β-Lg-Au NCs) could serve as a sensor for the detection and quantification of Hg2+ in beverages, urine, and serum with high sensitivity.