In this work, the interaction between nano-TiO(2) and trypsin was investigated, and the mechanisms of the interaction were explored by the methods of UV-vis detection, circular dichroism (CD), and fluorescence. The results clearly demonstrated that nano-TiO(2) had an inhibitory effect on the enzyme activity. The activity was decreased to 64% of the untreated trypsin in the presence of 300 μg/ml nano-TiO(2). UV spectrometry proved that nano-TiO(2) had a strong physical absorption effect on trypsin, and the CD spectra revealed that the secondary structure of trypsin was partly destroyed while bound together with nano-TiO(2). The ratio of α-helix increased from 7.9% to 12.8% in the presence of 100 μg/ml TiO(2) while the ratio of β-sheet decreased from 48.7% to 36.4%. Furthermore, the fluorescence spectrometry indicated that nano-TiO(2) could quench the intrinsic fluorescence of trypsin through static quenching. Meanwhile, the binding constant was calculated to be 1, and the process of binding of trypsin on nano-TiO(2) was a spontaneous molecular interaction procedure in which electrostatic interaction plays a major role. Our study was to provide a useful approach for evaluating the health risk of nanomaterials on level of proteins.