In this study, a supramolecular fluorescent material was constructed by using double-cavity twisted cucurbit[14]uril (tQ[14]) and positively charged Astrazon Pink FG (APFG) based on the non-covalent host-guest interaction for the first time. The thermodynamic parameters of the APFG@tQ[14] in aqueous solution were determined by isothermal titration calorimetry (ITC), the results indicated that the spontaneous assembly of APFG@tQ[14] is mainly driven by enthalpy. The intramolecular charge transfer (ICT) effect induced the APFG@tQ[14] probe to emit a strong orange-red fluorescence. Due to its good optical properties and low toxicity, APFG@tQ[14] was successfully applied to multi-channel imaging of HK2 cells. This demonstrates the great potential of APFG@tQ[14] in labeling and tracking living cells.