Aim. Mammalian tyrosyl tRNA synthetase (TyrRS) is composed of N-terminal catalytic miniTyrRS and non-catalytic C-terminal domain. After cleavage both domains of TyrRS reveal non-canonical cytokine functions. It is important to study the conformational changes of miniTyrRS in the course of ligands binding in different nanocomposite complexes. Fluorescence spectroscopy is a very powerful method to detect the local conformational changes of proteins. The study of single-tryptophan form of the protein can provide important information about flexibility and local conformational changes of the protein functional sites. Methods. Site-directed mutagenesis, bacterial expression, fluorescence spectroscopy. Results. Intrinsic fluorescence characteristics of single-tryptophan Trp40-mini TyrRS were measured, a spectral maximum at 332 nm was revealed, which corresponds to the buried state of Trp40 fluorophore in protein globule. Fluorescence quenching of Trp40 by acrylamide revealed the existence of conformational flexibility of mini TyrRS. Conclusions. Fluorescence studies of the single-tryptophan form of tyrosyl-tRNA synthetase revealed a buried state of Trp40 fluorophore but high conformational flexibility of the enzyme at the nanosecond time scale.