The synthesis of cationic amphiphilic pullulan having dimethylaminopropyl groups was performed in dimethyl sulfoxide using N, N′-carbonyldiimidazole as activator. The pullulan derivatives with three different substitution degrees were characterized by FTIR, 1H and 13C NMR, elemental analysis, and conductometric titrations. The aggregates characteristics were determined by steady-state fluorescence (critical aggregation concentration and polarity) and capillary viscometry measurements. Their behavior in aqueous dilute and semidilute solutions was investigated by viscosimetric experiments and fluorescence technique over the concentration range of 0.01–10 g dL−1, as a function of the degree of substitution with pendant groups. Intrinsic viscosity determined using Wolf equations provided information on the conformation of polymer chains. The results highlight the appearance of intramolecular and intermolecular aggregates and the dependence of their characteristics on the degree of substitution with pendant groups.