Transglutaminase (TGase) is an enzyme that catalyzes the post-translational covalent cross-linking of Gln-and Lys-containing peptides and/or proteins according to its substrate specificity. We have recently designed a variety of Gln-donor fluorescent substrates of microbial transglutaminase (MTG) from Streptomyces mobaraensis and evaluated their potential use in MTG-mediated covalent protein labeling. The newly designed substrates are based on the relatively broad substrate recognition of MTG for the substitution of the N-terminal group of a conventional TGase substrate, benzyloxycarbonyl-l-glutaminylglycine (Z-QG). It is revealed that MTG is capable of accepting a diverse range of fluorophores in place of the N-terminal moiety of Z-QG when linked via a suitable linker. Here, we show the potential utility of a new fluorescent substrate for peptide tag-directed covalent protein labeling by employing fluorescein-4-isothiocyanate-β-Ala-QG as a model Gln-donor substrate for MTG.