The molecular recognition of Cucurbit[6]uril (CB[6]) with Oxytetracycline (OTC) was studied by fluorescence spectra and UV spectra. The results showed that the fluorescence intensity and the UV absorbance of OTC were enhanced as host molecules were added, which showed that CB[6] and OTC were interacted. The inclusion of OTC was detected by fluorescence spectra and UV spectra with an apparent 1∶1 interaction, which was also observed through the Benesi-Hildebrand method, OTC can form 1∶1 complex with CB[6] under acid condition and it can not form complex with CB[6] under basic condition. The thermodynamic parameters (stability constants Ks, Gibbs free energy change ΔG, enthalpy change, and entropy change) for the complexation process were determined. The thermodynamic studies indicated that the inclusion reactions between CB[6] and OTC were the hydrophobic interaction and endothermic process.