Abstract: A novel fluorescent strategy is developed for the sensitive determination of trypsin by using DNA-templated Au nanoclusters (DNA-AuNCs) as fluorescent probes. In the presence of trypsin, bovine serum albumin is hydrolyzed to small peptides, releasing the free cysteine residues. A nonfluorescent coordination complex is formed by Au-S bond between cysteine residues and AuNCs, leading to obvious decreasement of the fluorescence intensity of AuNCs. The linear range of this method is range from 0.1 to 2.0 mg/L and with the limit of detection of 20 μg/L. The method has been applied for determination of trypsin in human serum samples.