The formation of the complexes of Norfloxacin with 2-methyl-β-cyclodextrin (Me-β-CD) was studied by UV-Vis absorption spectroscopy, fluorescence and nuclear magnetic resonance spectroscopy (NMR). Experimental conditions including Me-β-CD concentration and media acidity were investigated in detail at room temperature. The results suggest that Norfloxacin exists in three molecular forms in aqueous solution at different pH values, namely, the acidic form, the neutral form and the alkaline form). Me-β-CD was more suitable for inclusion of Norfloxacin in the acidic medium. The binding constant (K) of the inclusion complex was determined by fluorescence measurement, and the complexation ratio was determined as 1:1 in the concentration range used in this study. A mechanism was proposed to explain the inclusion process based on the experimental NMR data.