Non-natural amino acids are important tools for site-selective probing of peptide properties and interactions. Here, for the first time a fluorescent L-amino acid, exhibiting excited-state intramolecular proton transfer (ESIPT) and hydration-sensitive dual emission, was synthesized. It is an analogue of L-tryptophan bearing a slightly larger 2-(2-furyl)-3-hydroxychromone aromatic moiety instead of indole. This new amino acid was incorporated through solid phase synthesis into NC(11-55), the zinc finger domain of the HIV-1 nucleocapsid protein, that exhibits potent nucleic acid chaperone properties. It was substituted for the Trp37 and Ala30 residues, located in the distal finger motif and the linker between the fingers of NC(11-55), respectively. Though the highly conserved Trp37 residue plays a key role in NC(11-55) structure and activity, its substitution for the new fluorescent analogue preserved the folding, the nucleic acid binding and chaperone activity of the peptide, indicating that the new amino acid can conservatively substitute Trp residues. In the presence of oligonucleotides, the Trp37-substituted peptide, but not the Ala30 variant, showed strong changes of their dual emission corresponding to local dehydration. The results are in line with NMR data, suggesting that the fluorescent amino acid interacts similarly to Trp37 with the nucleic bases and is thus screened from water. Due to the exceptional sensitivity of its ESIPT fluorophore to hydration in highly polar environment, the new amino acid appears as a promising tool for substituting Trp residues and site-selectively investigating peptide-nucleic acid complexes.