Water-soluble β-cyclodextrin grafted with chitosan (CD-g-CS) was carried out by quaternizing the CD-g-CS with glycidyltrimethyl ammonium chloride (GTMAC) under mild acidic condition, corresponding to the quaternized CD-g-CS (QCD-g-CS). The degrees of substitution (DS) and quaternization (DQ), ranging from 5% to 23% and 66% to 80%, respectively, were determined by (1)H NMR spectroscopy. Self-aggregates formation of all QCD-g-CSs were investigated in water using dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM) techniques. The result revealed that all QCD-g-CSs are able to form self-aggregates in water. Large particle sizes ranged from 800 to 3000nm were obtained by DLS while zeta-potentials were ranging from 25 to 40mV. AFM and TEM depicted a spherical shape with particle sizes ranging from 100 to 900nm. Mucoadhesive and cytotoxic properties of all QCD-g-CSs were evaluated using a mucin particle method and MTT assay compared to quaternized chitosan (QCS). It was found that the mucoadhesive property increased with decreasing DS due to less quaternary ammonium moiety into the chitosan backbone. On the other hand, the cytotoxicity increased with increasing DS even though the DQ is decreased.