Conjugation of radiofluorinated prosthetic groups to primary amines of peptides in an aqueous medium is still considerably challenging. Herein, we report a one-pot cascade synthesis of 1-[18F]fluoro-4-isothiocyanatobenzene ([18F]2d), an isothiocyanate-functionalized prosthetic group for radiolabeling of various peptides in aqueous medium. The developed compound [18F]2d was synthesized in >99% radiochemical purity with 22.9 ± 3.8% (n = 12) decay-corrected yield having molar activity of 0.65 ± 0.19 (n = 12) GBq/μmol. Various clinically important peptides including prostate-specific membrane antigen vector, octreotide acetate, biotin analogue, Arg-Gly-Asp analogue, and bradykinin were successfully conjugated with [18F]2d in an aqueous medium in a good to moderate radiochemical yield. The overall synthesis of [18F]2d and its conjugation with a peptide take around 155 min, including purification.