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A new anthracene labeled pyridinium amide–urea conjugate 1 has been designed and synthesized. The receptor shows a different fluorometric response with L-N-acetylvaline and L-N-acetylalanine salts in CH3CN in contrast to the other salts of L-N-acetyl α-amino acids and (S)-α-hydroxy acids studied. Upon complexation of the tetrabutylammonium salt of L-N-acetylvaline, the emission of 1 increases accompanied by the formation of a new band at higher wavelength and this characteristic change distinguishes it from other anionic substrates studied. The binding interaction has been studied by 1H NMR, fluorescence and UV titration experiments.