In this study, the topographical characteristics of protein-based binders in ancient Chinese wall paintings and the properties of their sources were investigated and identified. Firstly, 15 samples of protein-based binders (skin glue, bone glue, egg glue, and milk glue) before and after ageing and mixing azurite were prepared. Next, the topographical characteristics, adhesive properties, and infrared spectra for each chemical group in the binders were analyzed by Atomic Force Microscopy (AFM) and Attenuated Total Reflection-Fourier Transform Infra-Red spectroscopy (ATR-FTIR), and the sources of various binders were identified by ATR-FTIR and Principal Component Analysis (PCA). Results showed that protein binders such as fish bladder, cow bone and egg white glue had lower roughness values and good consolidation properties. The most distinctive feature of skin and bone glues before and after ageing or mixed with azurite was the stretching vibration peak of CH2 deformation vibration at around 1335 cm–1, while the significant characteristics of egg and milk glue was the C=O stretching vibration in Serine at 1306 cm-1 and C=O stretching vibrations in aspartic acid and glutamic acid at 1416 cm-1. In addition, the significant differences of absorbance peaks were observed in the IR spectra of various bone, skin, egg, and milk glue samples before and after ageing while the spectral differences become insignificant after mixing with azurite. The results of PCA confirmed that egg yolk, ewe’s and cow’s milk were well differentiated from animal skin and bone glue regardless whether fresh, aged or mixed with azurite. In addition, the differences between skin and bone glues were significant, and the various types of bone glue could be also differentiated before and after ageing.