In the infrared spectroscopic study of aqueous protein solution, the characteristic peaks of solvent water and protein overlap partially or completely, which seriously affects the quantitative and structural analysis of protein. In this study, the solvent single beam spectra with arbitrary intensity were successfully synthesized by using two background samples. Therefore the solvent peaks in protein solution can be removed completely. The results of the hybrid spectrum were compared with those of the subtraction spectrum, and it was found that the hybrid spectrum has obvious advantages. The secondary structure information obtained from the second derivative of the hybrid spectra of BSA is in good agreement with that reported in the literature. Hybrid spectroscopy has also been used to study the thermal behavior of aqueous protein solutions. Without the interference of water peaks, the resolution of the amide Ⅰ and amide Ⅱ bands become easier. As the temperature increased, the peak position of the protein shifted. After high-temperature treatment, the bioactivity of BSA is lost.