Chitosan is one of the most prevalent biomass materials, and its physicochemical and biological characteristics, such as solubility, crystallinity, flocculation ability, biodegradability, and amino-related chemical processes, are directly connected to the degree of deacetylation (DD). However, the specifics about the effects of the DD on the characteristics of chitosan are still unclear up to now. In this work, atomic force microscopy-based single-molecule force spectroscopy was used to study the role of the DD in the single-molecule mechanics of chitosan. Even though the DD varies largely (17% ≤ DD ≤ 95%), the experimental results demonstrate that the chitosans exhibit the same natural (in nonane) and backbone (in dimethyl sulfoxide (DMSO)) single-chain elasticity. This suggests that chitosans have the same intra-chain hydrogen bond (H-bond) state in nonane and to which these H-bonds can be eliminated in DMSO. However, when the experiments are carried out in ethylene glycol (EG) and water, the single-chain mechanics are increased with the increases of the DD. The energy consumed to stretch chitosans in water is larger than that in EG, indicating that amino can form a strong interaction with water and induce the formation of the binding water around the sugar rings. The strong interaction between water and amino may be the key factor for the well solubility and chemical activity of chitosan. The results of this work are anticipated to provide fresh light on the significant role played by the DD and water in the structures and functions of chitosan at the single molecular level.