A hydrophobic carbon dots (Glc-OCDs) derived from octadecylamine and glucose were successfully syn-thesized for the first time and then grafted onto the porous silica surface by the “Nano-on-Micro” strat-egy,which was served as a new stationary phase (Sil-Glc-OCDs) for reversed-phase liquid chromatogra-phy.The structure of this stationary phase was carefully verified by laser scanning confocal microscope,Fourier transform infrared spectrometry,elemental analysis,contact angle measurement,etc.Several an-alytes including seven polycyclic aromatic hydrocarbons,eight alkylbenzenes,eight phenols and seven sulfonamides can be well separated on this stationary phase.Better separation performance for certain analytes over commercial C18 column was obtained.Interestingly,this stationary phase exhibited excel-lent chromatographic selectivity in the separation of the isomers of tert-butylbenzene,sec-butylbenzene,isobutylbenzene and n-butylbenzene.In addition,this new Sil-Glc-OCDs column was also applied for detection of calycosin-7-glucoside,ononin,calycosin,formononetin,genistein and isorhamnetin in the extract of Radix Astragali,which were found that the concentration was 0.15 g/L,0.088 g/L,0.14 g/L 0.086 g/L,0.18 g/L and 0.29 g/L,respectively.We believe that this CDs-grafted silica materials are promis-ing for chromatographic separation.