Abstract: Atomic Force Microscopy imaging is generally used for investigating the molecular structure of polysaccharides. However, for ionic polysaccharides, it is still difficult to obtain clear images with monolayer structure under the limited observation region based on the reported sample treatment methods. Taking xanthan gum as the typical ionic polysaccharide, in order to improve the image quality without changing the structure of polysaccharide, the sample preparation procedures were optimized, including sample concentration, variety and concentration of salt ions, and treatment time. The results showed that monolayer structure of xanthan was observed on clear mica when xanthan concentration was 1~5 μg/mL. However, the observed monolayer structure became much less when the observation region was 15 μm×15 μm. Then, the mica was treated and the treatment conditions were optimized. The optimized treatment conditions were selected as 2 min treatment by using 0.5 mmol/L calcium chloride solution or 0.5 min treatment by using 2 mmol/L calcium chloride solution. Under this condition, the molecular structure of 5 μg/mL xanthan gum was observed. The results illustrated that the number of monolayer xanthan gum molecular chains greatly increased within the observation region of 3 μm×3 μm, as compared with that on the untreated mica. Meanwhile, the treatment of mica could keep the height and morphology of monolayer structure of xanthan gum. In addition, the images of monolayer structure with more homogenous distribution could be obtained by increasing the drying temperature for mica. This study could provide an experimental reference for the sample preparation of other anionic polysaccharide in AFM imaging.