A series of chemiluminescent compounds containing a hydrazide group as a nucleophilic functional group has been synthesized. The syntheses were started from chemiluminescent luminol and isoluminol. The linker moiety was easily introduced onto non-nucleophilic exocyclic amino groups of luminol and isoluminol by gentle heating with cyclic acid anhydrides such as glutaric anhydride. The resulting carboxy group was converted to hydrazide by a simple condensation reaction using carbodiimide. Although majority of the synthesized compounds did not emit strong light, a sufficient chemiluminescence intensity was obtained from luminol-amido-C2-hydrazide ( L2H ) comprising of luminol scaffold with a dimethylene linker. The ability of L2H to form a covalent bond with a macromolecule was further investigated by incubation with oxidized horseradish peroxidase. The analysis on matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS revealed that the coupling efficiency of L2H was similar to that of commercially available labelling reagent having a hydrazide group. These results suggested that L2H , the luminol hydrazide containing a dimethylene linker, could be useful for the labelling of macromolecules in the sensitive bioassay such as chemiluminescence immunoassay.