Abstract: A determination method was established for the monosaccharide composition and content of tobacco water-soluble polysaccharides by 1-phenyl-3-methyl-5-imidazolinone (PMP) pre-column derivatization high performance liquid chromatography. Complete acid hydrolysis method was employed to hydrolyze tobacco water-soluble polysaccharides, and the hydrolysis products were subjected to PMP derivatization reaction. The signal of monosaccharide derivatives of tobacco water-soluble polysaccharides after acid hydrolysis was determined by reverse phase high-performance liquid chromatography. The method was used to explore the monosaccharide composition and content of tobacco water-soluble polysaccharides of different regions and types. The optimized detection conditions are as follows: 0.05 mol/L phosphate (KH₂PO₄-NaOH, pH 7.0) buffer (solvent A) and acetonitrile (solvent B) were chosen as the mobile phase of elution system at the isocratic elution condition (84:16), with Agilent Eclipse XDB-C18 column (250 mm×4.6 mm, 5 μm); the UV detection wavelength was 250 nm. Results showed that the linear relationship is good for the nine monosaccharides in the concentration range of 20.0-800 μg/mL, with the correlation coefficient (R²)>0.9927. The limits of detection and quantification were 0.07-0.17 and 0.22-0.56 μg/mL, respectively. The average recoveries ranged 82.3%-104.0 % in spiked samples. The tobacco water-soluble polysaccharides were composed of mannose (Man), rhamnose (Rha), galactose (Gal), arabinose (Ara), glucose (Glc), glucuronic acid (GlcUA), and galacturonic acid (GalUA), with Glc and GalUA accounting for a higher proportion. There were significant differences in the monosaccharide composition of tobacco water-soluble polysaccharides from various regions and types. The method is fast, simple, highly sensitive, and has good repeatability, thus can be used for monosaccharide composition analysis of tobacco polysaccharides. At the same time, it provides a basis for further research on the structure of tobacco polysaccharides.