Undesired adsorption of proteins brings big troubles to marine structures. The settled proteins change the physical and chemical properties of the surfaces, which allow marine fouling organisms to settle down on the structures. Therefore, to understand the adsorption mechanism of proteins is very helpful to find an environment-friendly solution against biofouling. Many approaches have been developed to study protein adsorption, but most of them are insufficient to give the chemical interaction information between proteins and surfaces. Fourier transform infrared spectroscopy with attenuated total reflection (FTIR-ATR) is an efficient, fast and non-destructive method for in situ surface measurement, which greatly minimizes the interference of water to infrared spectra, because of the very small depth of penetration of the evanescent wave. In this paper, an in situ FTIR-ATR technology was used to investigate the adsorption process of trypsin on a bare ZnSe surface and on a TiO2 coated ZnSe surface, and the effect of calcium cation strength and ultraviolet light irradiation on the secondary structure of trypsin were also evaluated. FTIR spectra of trypsin showed that Amide I band red shift and Amide Ⅱ band blue shift in aqueous environment on both surfaces compared with the dry trypsin powder, and the addition of calcium cations further changed the Amide bands position, which indicated that the change of the secondary structure could be interfered by the environment. The hydrogen bond formation between water and trypsin, the interaction between surface and trypsin, the interaction between hydrated calcium cations and trypsin, are major factors to change the secondary structure of trypsin, and UV light irradiation also showed its influence for the secondary structure.