A new type of amphiphilic chitosan, which was synthesized through the use of both hydrophilic carboxymethyl and hydrophobic hexanoyl substitutions, was employed to self-assemble into a hollow nanocapsule in an aqueous environment. Critical aggregation concentration (cac) and zeta potential were experimentally identified for the amphiphilic chitosan (CHC). Both experimental data suggested that the self-assembly behavior of the CHC is fundamentally determined as an interplay between the hydrophobic interaction and the variation of the zeta potential upon hexanoyl substitution, which further influenced the nanostructural evolution of the nanocapsules. Higher hexanoyl substitution promoted larger nanocapsules, ca. 200 nm in diameter, while a reduced zeta potential was correspondingly detected, and vice versa, forming smaller nanocapsules, ca. 20 nm in diameter. The self-assemble mechanism, together with the corresponding nanostructural stability, of this unique CHC nanocapsule was also proposed in terms of intermolecular interaction and thermodynamic reason. By taking the advantage of the self-assemble (or self-aggregation) capability, the CHC was employed for drug encapsulation, i.e., doxorubicin, an anticancer molecule; we found in this preliminary evaluation that it reached an efficiency of 46.8%, and a corresponding drug release from the nanocapsules for a time period exceeded 7 days can be achieved in vitro.