The murals in Prince Shis Palace are typical of the murals in southern China, and they are also the most complete and painted mural relics of the Taiping Heavenly Kingdom. Because it is located in a humid area in the south, under the influence of environmental factors, the murals of Prince Shis Palace have appeared with various diseases, and the preservation status is not optimistic. In order to protect this important mural, it is necessary to conduct scientific research on the production materials of murals. The nondestructive analysis was carried out by p-XRF and Raman spectroscopy. It was found that cinnabar (HgS) and hematite (Fe2O3) were used as the red pigments for the murals, and the white pigments were mainly calcite (CaCO3). Analytical conditions limit other pigments, and no color components are detected. Several trace pigment and ground samples were obtained by sampling, and spectroscopic analysis techniques such as microscopic infrared spectroscopy (μ-FTIR), X-ray diffraction (XRD), micro-Raman spectroscopy (μ-Raman) and pyrolysis-gas chromatography/mass spectrum(Py-GC/MS) were used in the laboratory to analyze the blue-green pigments, ground components, binding material, types of fibers, etc. The results show that blue pigment is the first modern synthetic pigment prussianblue (Fe4［Fe(CN)6］3), introduced into China and widely used in the middle and late Qianlong period. There are three kinds of green pigments, including synthetic atacamite, lavendulan, green discoloration product of paris green, and cumengite, first discovered in China. However, the current research has not seen the case of the use of cumengite as a pigment, and there is no report of such minerals in China. It is believed that the cumengite found in the murals is the product of the discoloration of other copper containing pigments. The results of Py-GC/MS show that there are proline and hydroxyproline in the pigment layer of the murals in prince Shis Palace, which are the main components of animal glue, so animal glue is used as the cementing material for the murals. By calculating the infrared spectral characteristic peak ratios (I1 595/I1 105) and R2 (I1 595/I2 900) of the fibers in the mural mud ground and lime ground, it is concluded that the fibers in the mural mud ground are ramie fibers and the fibers in the lime ground are cotton fibers. The research provides a scientific basis for pigment identification, restoration materials selection and scientific conservation of murals in Prince Shis Palace of the Taiping Heavenly Kingdom.