Isothiocyanatoluminol (ITL) was successfully synthesized from the reaction between luminol and thiophosgene in the presence of triethylamine. The optimized conditions were extended for the preparation of isothiocyanatoisoluminol (ITIL) and phenyl isothiocyanate (PI). The chemiluminescence (CL) of ITL was comparable to luminol. ITL was used as a CL labeling reagent for amino acid and bovine serum albumin (BSA) in which the -NH2 terminus of amino acid residue present in BSA acts as the labeling site.