The complexation of kaempferol with cyclodextrins (CDs) (β-, G2-β-, and HP-β-CDs) in basic medium was studied, and the complexation constants (Kc) were calculated by using enzymatic, solubility, and fluorometric methods. This is the first time that a decrease in fluorescence has been observed as result of the analyte complexation by CDs. The highest Kc value for kaempferol complexation was obtained for HP-β-CDs. To establish the validity of the fluorometric method for determining the Kc between kaempferol and CDs, the same parameters were also determined by enzymatic and solubility methods. The enzymatic method was carried out by using horseradish peroxidase as oxidative enzyme, and the Kc values obtained were similar to those obtained by using the solubility method. However, the fluorometric method underestimated the Kc value by about 1.2-fold with respect to the other methods used. In all cases HP-β-CDs showed the highest Kc value, indicating that they are more efficient in the formation of inclusion complexes with kaempferol.