Leather artifacts hold significant historical and cultural value in human civilization. During long-term preservation, ancient relics, especially waterlogged leather artifacts, are susceptible to protein degradation. Therefore, analyses of the structure and protein composition of these ancient relics are crucial for their effective conservation. However, comprehensive research in this field is scarce and urgently needed. In this study, systematic investigations of the structures of fresh vegetable-tanned leather, dried leather artifacts, and waterlogged leather artifacts were performed from multiple perspectives. Compared with fresh vegetable-tanned leather and dry leather artifacts, the deterioration of waterlogged leather artifacts resulted in a darkened color, increased brittleness, and reduced fiber structure. Infrared analyses revealed that the characteristic peaks of the amide II and III bands were not present in waterlogged leather artifacts. The species of the leather artifacts were analyzed using enzyme-linked immunosorbent assay (ELISA). Furthermore, comparative proteomics analysis reveals that protein species identified in waterlogged leather artifacts were significantly lower than in fresh vegetable-tanned leather. Type I collagen, along with many structural proteins, was degraded. The degradation of collagen and other structural proteins, along with fiber destruction, caused severe morphological deterioration of waterlogged leather artifacts. A strategy combining species identification by ELISA and proteomics allows the identification of leather proteins with unprecedented levels of speed, sensitivity, and specificity.