Abstract: Water-soluble gold nanoclusters (T-Au NCs) with red fluorescence were synthesized with trypsin as stabilizing and reducing agent in the mixed solution of trypsin and chloroauric acid. The optimum synthesis conditions for T-Au NCs were defined by investigating the reaction time and the mass concentration of trypsin solution. Fluorescence spectroscopy characterization indicated the maximum excitation and emission wavelengths of the prepared T-Au NCs were 500 nm and 640 nm respectively. The valence state of gold in T-Au NCs was estimated by X-ray photoelectron spectroscopy. The fluorescence quantum yield of T-Au NCs was 2.2%. Based on the fluorescence quenching effect of H2O2 to T-Au NCs, H2O2 residues in the food were determined by fluorescence spectroscopy. The linear range of H2O2 was 0.050-1.00 mmol·L-1, with detection limit of 7 μmol·L-1. The determination results of H2O2 in 4 kinds of food were in consistent with those obtained by standard method.