Polyelectrolyte complexes (PECs) of hyaluronic acid (HA) and diethylaminoethyl dextran (DEAE-D), promising for biomedical applications, have been investigated with respect to physicochemical properties, mainly in terms of particle size and relative hydrophobicity as well as storage stability. Influences of charge ratio, polymer concentration, molar masses of HA, ionic strength and mixing methods have been particularly investigated. The complexation between HA and DEAED generally resulted in colloidal particles having mean hydrodynamic diameter of 150–350 nm, with larger particle size observed at negative to positive charge ratio (n-/n+) further from the unity. Higher polymer concentration or higher molar mass of HA also led to higher particle size of the PECs, whereas the increasing ionic strength led to a non-monotonic evolution in particle size. The effects of mixing mode and mixing order on particle size were interdependent and depended also on n-/n+. PECs at n-/n+ ≤ 0.4 showed lack of stability, which seemed not to be sensitive to storage temperature but significantly improved at higher salt concentration or lower polymer concentration. The relative hydrophobicity of such PECs was also confirmed by the fluorescence spectra of pyrene incorporated in PEC particles. Such results gave a better insight to polysaccharides-based PECs, especially as a potential system to encapsulate hydrophobic active molecules for drug delivery purposes.