Leather cultural relics, made of animal skins, are treasures left by human beings in the process of understanding and transforming nature. These relics are meaningful historical materials that could be used to study the ancient society and history. Leather is a natural organic polymer material, the main component of which—collagen—is susceptible to denaturation and deterioration depending on the environment of its preservation. Therefore, it is of great significance to study the microstructure of collagen for understanding the deterioration of ancient leather. Hydrogen-deuterium exchange (HDX) technology is often used to study protein structures based on the principle that the unstable hydrogen atoms in proteins are exchanged with deuterium atoms, but its application to the study of the structure of ancient leather collagen has not been reported. In our study, HDX technology and Fourier transform infrared (FTIR) spectrometry were used to analyze and compare the triple helix structure of the collagen in modern, artificially-aged and ancient leather. The changes of the collagen microstructure were characterized by the position of the characteristic absorption peak and the deuteration rate. The results show that the triple helix structure in modern leather collagen is intact, and it is difficult for deuterium atoms to enter and be exchanged with amino hydrogen atoms in the main chain, with the deuteration rate being 32.09%. However, during the deterioration of artificially-aged and ancient leather, the solvent accessibility of the amide hydrogen in the main chain of collagen increased, the hydrogen bond formed by the amide hydrogen changed, and the hydrogen bond maintaining the stable triple helix structure was broken. The triple helix structure was disintegrated to a certain extent and glycine at the center of the helix structure was exposed, increasing the solvent accessibility of the amide hydrogen in the main chain and the triple helix structure became loose and unstable. The hydrogen-deuterium exchange rate was accelerated and the hydrogen-deuterium exchangereaction was more likely to occur. Among the three ancient leather samples, the deuteration rate of old3 is 65.87%, which was the lowest, and the triple helix structure in old3 is the least damaged and the least deteriorated. Compared with the previously reported literature about the research on protein structure by hydrogen-deuterium exchange, our study expands in the following aspects:First, it is pointed out that the amino hydrogen in the main chain, especially the amide hydrogen of glycine, is the main object of exchange with deuterium atoms through the analysis of the characteristic structure of collagen; Second, the degree of hydrogen-deuterium exchange is quantified by the position of the characteristic peak and the deuteration rate according to the characteristic that the change of the mass number of corresponding groups after hydrogen-deuterium exchange will lead to the change of stretching vibration frequency, and the amide A band of collagen in Fourier transform infrared spectrometry is selected as the characterization tool of hydrogen-deuterium exchange reaction. The combination of HDX technology and FTIR spectrometry are introduced into our study of the collagen structure of leather cultural relics, which contributes to the deep understanding of the deterioration mechanism of leather cultural relics. It provides a new method and thought for the study of the collagen microstructure and the analysis of the deterioration degree of leather cultural relics.