Small molecules, peptides, and proteins can be imprinted using mixtures of organic silanes. Molecular imprints may serve as artificial receptors,i.e., biosensor sensing elements for detection of chemical and biological toxins, drugs, and environmental hazards. One method for detection of imprint-bound molecules is fluorescence. Molecular imprints to N-acetyltryptophanamide (NATA) and fluorescein were prepared, and their respective binding constants determined using steady-state fluorescence spectroscopy. Stern-Volmer fluorescence quenching plots of imprint-bound molecules using potassium iodide (KI) and acrylamide indicate bound molecules are shielded from the solvent environment. Scatchard plot analysis revealed two binding affinities,i.e., aKd= 0.13 and 2.5 μM for NATA binding to NATA imprints. Interestingly, NATA exhibited higher affinity,i.e., Kd= 1.3 and 35 nM, for the fluorescein imprint. These data support the usefulness of fluorescence techniques in molecular imprint-based detection technology.