In this study two types of water-soluble βCD grafted chitosan were synthesized and compared based on similar degree of N-substitution of βCD moiety; QCD23-g-CS contained methylene spacer and QCDCA22-g-CS contained citric acid spacer. The QCD23-g-CS demonstrated greater eugenol (EG) encapsulation efficiency than that of QCDCA22-g-CS. The micelle-like assemblies of QCD23-g-CS led to slower release of EG while it did not observe in case of QCDCA22-g-CS. It was found that EG could absorb on chitosan backbone according to in silico modeling. Cytotoxicity of both derivatives against buccal mucosa cell is concentration-dependent. The QCDCA22-g-CS demonstrated stronger mucoadhesive response than that of QCD23-g-CS, due to hydrogen bonding according to mucin particle and SPR methods. Our results revealed that the spacer on both derivatives played an important role on binding affinity with EG, releasing profile and mucoadhesive property. These derivatives could be considered as promising carriers for mucosal delivery system.