Murals are an important decorative element of temple architecture and an important component of Tibetan Buddhism art. Tashilhunpo Monastery was built in the 12th year of the reign of Ming Emperor Zhengtong (1447AD), which treasures unique and resplendent paintings that plays an important role in Tibet. It is the largest temple in later Tibet, belonging to the Gelug sect of Tibetan Buddhism. Tashilhunpo Monastery has been serving for spreading Buddhist culture since its establishment and has undergone frequent large-scale construction. Investigating the painting materials and techniques now becomes one important part of cultural heritage protection. A total of 8 samples were collected from the typical murals on the north wall of the fourth floor of Maitreya Hall and the west side of the South Hall of Exoteric Buddhist Seminary. Pigments, the ground layer and the inside structure of the painting were studied by three-dimensional video Microscopy, scanning electron microscopy in combination with energy dispersive X-ray microanalysis, polarizing microscope, X-Ray Diffraction, microscope and laser Raman technique. The results show that three layers of the wall painting cross-section correspond to a ground layer, a yellow preparation layer, and a paint layer. Natural and synthetic pigments are both used in the paintings, including cinnabar (HgS), orpiment (As₂O₃), charcoal (C), antlerite (Cu₃(SO₄)(OH₄)), C. I. Pigment Red 14 (C₂₄H₁₇ClN₄O₄), synthetic ultramarine blue (Na₈(Al₆Si₆O₂₄)Sn), Phthalocyanine green G (C₃₂H₃Cl₁₃CuN₈-C₃₂HCl₁₅CuN₈). Pigment red 14 and phthalocyanine green are organic synthetic pigments, while synthetic ultramarine blue is inorganic synthetic pigments. As a mineral pigment, antlerite has been used in easel paintings, murals, manuscripts and other artistic works in Europe, but the history of usage has not been found in China. This discovery expands the knowledge of green pigments.The study demonstrated that loess and aga soil was the base of the wall paintings and painted with kinds of color finally according to the religious ritual. Furthermore, the research findings show traditional materials for Tibetan murals and modern synthetic materials, indicating that several wall paintings have been repaired or repainted later. The results make up for the murals research vacancy of Tashilhunpo Monastery and provide important evidence for the complement and improvement of its repair history.