Samye Monastery is known as the first temple in Xizang, and a large number of murals are preserved therein. In this study, X-ray diffraction, scanning electron microscopy and optical microscopy were used to analyze the structure and material composition of the mural plaster layer of Samye Monastery. The results show that the main components of the mural plaster layer were Aga soil, sand and charcoal, with a mass ratio of about 24∶75∶1. Charcoal is not a necessary material for making Tibetan murals, but why was the charcoal added to the murals in Samye Monastery? Through infrared thermographic inspection, it was found that the mural dado was wet, and the addition of charcoal into the plaster layer could play a moisture-proof effect. This study could provide reference for the conservation and restoration of the murals in Samye Monastery.