<p>The Cookson reagent (4-phenyl-1,2,4-triazoline-3,5-dione, PTAD) is a powerful dienophile for the Diels–Alder reaction and is used as a protecting group for conjugated dienes in organic chemistry. PTAD also undergoes a selective ene reaction with tyrosine (Tyr) residues in aqueous solvents. In this study, we investigated the ene reaction products with Tyr using two Cookson-type fluorescent reagents, 4-(1-naphthyl)-1,2,4-triazoline-3,5-dione (NTAD) and 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQTAD). The fluorescence properties were examined to develop peptide probes using Tyr itself, angiotensin II, and amyloid β₄₀ (Aβ₄₀) as models. NTAD-Tyr showed major changes in fluorescence spectra based on the conditions of the surrounding environment, such as pH, solvent hydrophobicity, and human serum albumin concentration. The increase in the fluorescence intensity of NTAD-Tyr was greater than that of DMEQTAD-Tyr, especially for pH and human serum albumin. Furthermore, a wavelength shift (blue shift) was observed for NTAD-Tyr but not for DMEQTAD-Tyr. Therefore, NTAD was used for the following peptide experiments. NTAD-Aβ₄₀ showed an unexpected red shift in a hydrophobic solvent, which was the opposite shift to NTAD-Tyr and NTAD-Ang II. In the aggregation study of Aβ₄₀ co-incubated with NTAD-Aβ₄₀, the fluorescence of NTAD-Aβ₄₀ increased and reached a plateau earlier than the common thioflavin T method for recognizing β-sheet formation.</p> Graphical Abstract <p></p>