Palm leaf manuscriptsarea kind of the oldestliterature with significant historical and literary values. The leaves are easilydamagedby external factors which can result in diseases. Water stainsareone of the most common diseaseson palm leaf manuscripts. They are permeable contamination depositsthatcan affect the appearance of palm leafmanuscriptsand even damage the subjects' stability. In this paper, visible-reflected images andultravioletfluorescence images oftwo sets of palm leaf manuscript collectionsare captured. Physical parametersincluding pH values and chromatic aberration in water-stain and non-water-stain areasare measured by portable pH meter and colorimeter. The results show that “tideline” is more obvious and water-stain areasareeasier to distinguish under ultraviolet light. Meanwhile, pHvalues are all smaller in water-stain areasand the color differencebetween water-stain and non-water-stain areas is obvious. The results of micro-X-ray fluorescence and Fourier transform infrared spectroscopy coupled with ATR show that elements including K, Ca, P, S, and Mn are enriched in water-stain areas, with the decrease of the relative contents of cellulose and hemicellulose. Acomprehensive determination methodof water stain on palm leaf manuscripts based on nondestructive techniquesis first establishedin this study, whichprovides a deep understanding ofwater stain pollution. The method established in this study is meaningfulfor the scientific and rational preservation of palm leaf manuscriptsand can be a direction for furthercleaning and removal of water stain pollution.