Bovine serum albumin (BSA) and glutathione (GSH) have been used to prepare gold nanoclusters (BSA/GSH-Au NCs) with excitation/emission wavelengths of 330/650nm from Au3+ at 70°C for 30min. In the preparation of BSA/GSH-Au NCs, BSA and GSH act mainly as capping and reducing agents, respectively. With the assistance of GSH, only 30μM BSA is required to prepare stable and bright BSA/GSH-Au NCs. In addition to BSA, small amounts of lysozyme, ovalbumin, and transferrin have also been used separately to prepare protein/GSH-Au NCs. The decreased order of the photoluminescence (PL) intensity of protein/GSH-Au NCs agrees with the decreased orders of size and number of tyrosine/cysteine of the four proteins. The PL of BSA/GSH-Au NCs is quenched by nitrite (NO2−) at pH 3.0, mainly due to the oxidation of BSA/GSH-Au NCs with nitrosyl ions (NO+). This approach allows detection of NO2− down to 0.3μM, with great selectivity and stability against salt (up to 500mM NaCl). Practicality of this simple and sensitive approach has been validated by the analysis of pond water samples.