The fluorescence quenching of cucurbituril-palmatine(CB-PAL) complexes by cetylpyridinium chloride (CPC) has been studied in aqueous solutions. The results show that the rate of the fluorescence quenching was proportional to the concentration of CPC. A fluorescent probe method of simply, accurately and high sensitivity was developed for the determination of CPC in pharmaceutical preparations and human urine through the supramolecular complex formation with CB. The linear range of the method was from 0.019 μg mL−1 to 3.556 μg mL−1, with a detection limit of 0.007 μg mL−1. Molecular modelling of the interaction between CPC and CB was established and was also confirmed by 1 H NMR. This paper also discusses the mechanism of fluorescence for the indicator probes. This shows that the proposed method has the potential for therapeutic monitoring, pharmacokinetics and clinical applications.