The interaction between 2-(2-Amino-3-Pyridyl)benzimidazole (2A3PyBI) and Cucurbit[6]uril (CB6) have been investigated in aqueous solution by using fluorescence and UV–Vis spectroscopy.The experiment was explored at four different selected pH environment which are pH=3.0,5.0,6.3 and 10.0.The results implies that 2A3PyBI has a dual emission fluorescence corresponding to the two proton transfer molecular structure (amimo and imino form) at the four selected pH values.The blue emission band increase apparently but the red emission band changed weakly in the present of CB6 indicates that encapsulation into the CB6 macrocycle cavity retargets the deprotonation process of the excited-stated form,and hence reduce its subsequent conversion to the imino form.The interaction ratio was found to be 1:1 by Benesi-Hildebrand method,and the maximum binding constant (Ka) was caculated to be 1.8×104 at pH=5.0 and the minimum binding constant to be 5×102 at pH=10.0,it indicates that the cationic form binds stronger with the CB6 macrocycle hydrophobic cavity than the neutral form.The caculated nonpositive Gibbs free energy (ΔG) indicated the process of 2A3PyBI encapsulates into the CB6 macrocycle hydrophobic cavity is spontaneous.