This research sheds light on the analysis of pigments and adhesives applied on a color painting on wooden architecture in Taiping Heavenly Kingdom Prince Dai’s mansion, located in Changzhou, Jiangsu Province in China. Four samples were collected from the painting above the building beam in the mansion, and the samples were analyzed and identified using a series of techniques, including polarized light microscopy (PLM), scanning electron microscope coupled with an energy-dispersive X-ray spectroscopy (SEM-EDS), micro-Raman spectroscopy (m-RS) and Fourier-transform infrared spectroscopy (FTIR). The results indicate that the red, black, blue, and green pigments were identified to be cinnabar, ivory black, indigo, and phthalocyanine green, respectively. The green pigment was inferred to be a lately repainted pigment based on its production age, suggesting that this ancient building had been refurbished or repaired. Given the good stability and visual effect of this green pigment, it is suggested to be used in future conservation processes. The pyrolysis-gas chromatography/mass Spectrometry (Py-Gc/Ms) results indicate that glue containing protein was used as a binder for the pigment samples, and that walnut oil might have been applied to the wooden architecture as a primer before painting. Our findings can well inform curators and conservators of the selection of appropriate restoration materials if necessary, and also provide data support for conservation of similar ancient buildings in southern China.