Carbon quantum dots (CQDs) were synthesized from brominated 1-hexadecyl-3-vinylimidazolium and citric acid, and a mixed-mode chromatographic (MMC) stationary phase, which can be used in the RPLC/HILIC/IEC chromatographic modes with a good separation efficiency, was successfully synthesized by click-chemistry bonding to a sulfhydryl-modified silica surface. The successful synthesis of the chromatographic stationary phase was demonstrated by its characterization. The effects of organic phase ratio, column temperature and buffer salts on the column were investigated, established the van't Hoff equation, and verified the ion-exchange effect of the column by separating benzoic acid and phenolic compounds under different pH conditions. The reversed-phase performance of the column was characterized by Tanaka test. Compounds such as alkylbenzenes, polycyclic aromatic hydrocarbons, and nucleosides can all be separated on the column in a short period of time. The reproducibility and stability of the columns were verified under the corresponding chromatographic conditions, and the results showed good reproducibility and stability of the columns. The chromatographic column was used to separate the base nucleosides in apples, and four base nucleosides were successfully isolated from apples.