In this study, an integrated analytical method was developed to investigate the composition of both the inorganic pigments and organic binders of polychrome relics in Maiji Mountain Grottoes in northwestern China. Cross-sections of each sample were prepared at the beginning of the study, and all experiments were carried out on these cross-sections. Polychromic structures were revealed by optical microscopy and scanning electron microscopy-backscattered electron imaging. Inorganic materials were determined by using SEM coupled with an energy dispersive spectrometer and μ-Raman spectrometer, whereas organic materials were identified by staining techniques and highly sensitive and specific immunofluorescence microscopy. Data showed that the red colors are attributed to one or two pigments of red ochre, cinnabar, and minium; the blue pigment is natural lazurite; the green pigment is ascribed to atacamite; the white color is attributed to potassium feldspar; and the black surface is formed by the discoloration of minium to plattnerite under the influence of environmental factors. Regarding organic binders used in painting and preparation layers, mammalian animal glue and chicken egg white were both found alone or in mixture. Finally, the conclusion is made that the Secco technique is employed in polychrome relics from Maiji Mountain Grottoes.