A novel ratiometric fluorescent sensor 1 having the 4-(N,N-dimethylamino)benzoate (DMAB) group as a twisted intramolecular charge transfer (TICT) dual fluorescence site and the guanidinium group as a binding site for an amino acid at the 12- and 3-positions of cholic acid, respectively, was designed and synthesized. The sensor 1 showed characteristic dual fluorescence and it was shown that the guanidinium group in the proximity of the DMAB group influenced largely the TICT formation process in 1. Liquid/liquid extraction experiments demonstrated that the intensity ratio of the TICT fluorescence to the locally-excited (LE) fluorescence (I(TICT)/I(LE)) observed from the organic layer decreased with an increase in the concentration of N-acetyl-d-phenylalanine (AcPhe) in the water phase. Such fluorescence titration experiments afforded the apparent binding constant between 1 and AcPhe to be 7.0 x 10(5) M(-1). Temperature dependence of the fluorescence spectrum of 1 in the absence and presence of AcPhe indicated that the primary origin of the change in the I(TICT)/I(LE) value upon recognition of the amino acid was the increase in the activation energy of the TICT formation process in 1.