The assembly of colloidal hyaluronic acid (HyA, as a polysaccharide) based hydrogel particles in an aqueous medium is characterized in the present paper, with an emphasis on the particular case of nanohydrogels formed by surfactant-neutralized polysaccharide networks. The structural changes and particle formation process of polysaccharide- and cationic-surfactant-containing systems were induced by the charge neutralization ability and the hydrophobic interactions of cetyltrimethylammonium bromide (CTAB) under different conditions. Based on the rheological, light scattering, ζ-potential, turbidity, and charge titration measurements, it can be concluded that the preparation of the HyA-CTAB particles can be greatly controlled. The results indicate that more available negative charges can be detected on the polymer chain at smaller initial amounts of HyA (c < 0.10 mg/mL), where a molecular solution can be formed. The change in the pH has a negligible effect on the formation process (particle aggregation appears at n/n~1.0 in every case), while the temperature dependence of the critical micelle concentration (c.m.c.) of CTAB determines the complete neutralization of the forming nanohydrogels. The results of our measurements confirm that after the appearance of stable colloidal particles, a structural change and aggregation of the polymer particles take place, and finally the complete charge neutralization of the system occurs.