The self-aggregation of polyelectrolytes having N-alkyl-N,N-dimethyl-N-(2-hydroxypropyl)ammonium chloride pendant groups (alkyl = octyl, dodecyl, or cetyl) randomly distributed along a polysaccharide backbone (dextran) was studied by steady-state fluorescence techniques using several free fluorescent probes or pyrene-labeled polymers and by viscometry. The onset, offset, and highest values of the fluorescence response of N-phenylnaphthylamine (NPN), pyrene (Py), and 1,6-diphenyl-3,5,6-hexatriene (DPH) were corroborated with NPN and DPH anisotropy and quenching experiments to describe the dynamic of hydrophobic microdomain formation and microdomain characteristics. The start of the aggregation process (critical aggregation concentration, cac) and the microdomain characteristics such as polarity, microviscosity, size, and number strongly depend on the alkyl chain length and the degree of substitution with cationic pendant groups. Fluorescence experiments with pyrene-labeled polymers and viscosity data suggest that microdomains are mainly formed by intramolecular hydrophobic associations, except for the polymers carrying octyl groups, where some intermolecular associations were revealed.