In this study, a new oxazolone derivative 4-{N,N-bis[2-phenyl-4-benzylidene-1,3-oxazol-5(4H)-one]amino}benzaldehyde (PB3) was synthesized and investigated as a fluorescent dye. The spectroscopic properties in different solvents were thoroughly studied. The experimental data were supported by quantum-chemical calculations using density functional theory. Measurements and theoretical calculations showed that the PB3 dye is characterized by non-monotonic solvatochromism, a strongly polar charge transfer excited state, a large Stokes’ shift, a high fluorescence quantum yield and a high fluorescence lifetime. Bioconjugate complexes (PB3–concanavalin A) were studied by circular dichroism (CD) spectroscopy. The results showed that the secondary structure of concanavalin A was not significantly influenced by the PB3-fluorophore. Conventional fluorescence microscopy imaging of Candida albicans cells, incubated with the PB3–concanavalin A, was demonstrated. The results from cytochemistry experiments demonstrate that the PB3 dye has valuable advantages compared to the other long-wavelength dyes in typical fluorescence-based cell labeling applications. In vitro tolerance was evaluated by the MTT method in the human colon adenocarcinoma cell line HT29. The PB3 and bioconjugate complexes (PB3–concanavalin A), in the range of concentrations tested, were not considerably toxic. The AutoDock simulations showed LYS46 as the most likely active site for covalent bond formation during PB3–concanavalin A conjugation. In addition, theoretical studies have shown that PB3 is characterized by good bioavailability and absorption/transmission across the cell membrane. This molecule will not bioaccumulate in living organisms and should be excreted in urine without interacting with other drugs. This work provided promising results for the red fluorescent probe (PB3) as a valuable alternative to commercial probes designed for cellular labeling in biological and biomedical research.