To regulate the sodium chloride content in Jinhua ham, the impact of NaCl on the activity and conformation of cathepsin B was investigated using spectroscopy and computational methods. The results showed that the activity of cathepsin B decreased with an increase in Na+ cation content and temperature. Additionally, decreased α-helix content and increased β-sheet content were observed. The increase in sulfhydryl group content was attributed to the breaking of original disulfide bonds in the molecular structure or the release of embedded groups. Furthermore, the surface hydrophobicity gradually declined, which was consistent with the analysis of endogenous fluorescence spectroscopy. At the molecular level, the number of hydrogen bonds formed in NaCl-treated samples decreased, and the interactions between the hydrogen bonding were less powerful, which caused instability in the binding of the protein and substrate. The conformation of cathepsin B accurately characterized its activity, and the structural changes had a macroscopic effect on the decrease in protease activity.