In this study, the materials and microstructure of ancient Chinese wall paintings from the Song-Jin period Xianqing temple were investigated using optical microscopy, scanning electron microscopy with energy dispersive spectroscopy, Raman spectroscopy, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, and pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS). Apart from comparing and analyzing the microstructures, materials, and painting styles of the wall paintings, radiocarbon accelerator mass spectrometry (AMS), in particular bomb pulse dating, was employed for the first time to determine the dating of the ancient Chinese wall paintings. Morphological results showed that the wall paintings on the east and west walls comprised a pigment layer, an underpainting layer made by chalk, and a soil layer. Raman spectroscopy confirmed that the pigments used included lazurite, green earth, massicot, amorphous carbon black, cinnabar, and chalk. In addition, a comparative analysis of the wall paintings with significantly different painting styles revealed that the wall paintings had not been repainted. The analytical results of radiocarbon content obtained by AMS revealed that the soil layers of the wall paintings were made in the Ming Dynasty (1368 to1644 AD), while the dating results of the underpainting layer further confirmed that the ancient wall paintings might be painted in 1963 AD or 1968 to 1972 AD. Microscopic and ATR-FTIR analyses further confirmed that the fibers detected in the underpainting layers of the wall paintings contained cotton. Based on the Py-GC/MS analysis, the presence of hydrocarbon compounds provided evidence of wax being used as binders in the wall paintings.