In this work, it was first found that tectoridin can bind to the fat mass and obesity-associated protein (FTO) by isothermal titration calorimetry (ITC). Spontaneous exothermic reactions occurred between tectoridin and FTO driven by entropy. A static quenching mechanism was developed for this interaction by using the spectroscopic technique. Results of both ITC and fluorescence spectroscopy indicated that hydrophobic interactions are predominant in the formation of the complex. The binding parameters obtained from ITC are comparable to those obtained from fluorescence spectroscopy. The inhibition of tectoridin on the demethylase activity of FTO was confirmed by enzymatic activity assays. This study will provide the basis for the development of inhibitors for FTO demethylase.