O-Carboxymethyl chitosan-functionalized multi-walled carbon nanotubes (MWCNTs/OMCS) were found to be a novel additive for high-speed counter-current chromatography (HSCCC). It was shown that a kind of liquid–liquid/solid (LLS) three-phase HSCCC system can be established with MWCNTs/OMCS as additive, and in this HSCCC system liquid–liquid partition chromatography and liquid–solid adsorption chromatography were combined. This study revealed that MWCNTs/OMCS can obviously improve peak resolution (R_s). Three flavonol aglycons (quercetin, luteolin and kaempferol) were used as model analytes to study the mechanisms of MWCNTs/OMCS as HSCCC solvent additive to improve R_s. It was indicated that MWCNTs/OMCS improved R_s not by increasing the stationary phase retention but by introducing intermolecular forces: electrostatic interaction, hydrogen bonding interaction and π–π conjugation.