We designed 6-dimethylamino 3-methyleneisoindolin-1-one as an environment-sensitive fluorophore, examining its applications for protein labeling. Synthesized 3-methyleneisoindolin-1-one exhibits solvatochromic fluorescence (λemmax; 472 nm in 2-PrOH, 512 nm in H2O). A positive linear dependence between λemmax and solvent dielectric constant (DC), as well as between Stokes shift and DC, and a negative correlation between fluorescence quantum yield and DC are observed in protic solvents. These properties are similar to those of the oxygen isosteric fluorophore, 4-dimethylaminophthalimide, a slovatochromic fluorophore utilized for labeling oligodeoxynucleotides (ODNs) and peptides. Notably, fluorescence intensity of 3-methyleneisoindolin-1-one is higher than the phthalimide in protic solvents used in this study. The 3-methyleneisoindolin-1-one demonstrated the higher stability in pH 8 solution than in pH 6 solution in contrast to the stability profile of the phthalimide, which was stable at pH 6 but was hydrolyzed at pH 8. We also synthesized an o-keto benzaldehyde derivative that converts a primary amine to 6-dimethylamino 3-methyleneisoindolin-1-one under biocompatible conditions and introduced it into ODNs for turn-on fluorescent protein labeling. The synthesized ODN with a protein-binding sequence of Escherichia coli DnaA was employed to modify the DNA-binding domain of DnaA, and the fluorescent properties of the modified protein were investigated.