Functional luminol derivative with cholesteryl substituted group has been designed and synthesized from the reaction of the corresponding precursor cholesteryl chloroformate with luminol. This compound can be spread on water surface to form stable monolayer. It has been found that UV and IR spectra confirmed the characteristic aromatic segment, imide group and cholesteryl substituted group. In addition, the CD spectra also showed positive CD signals, which may be attributed to the chiral cholesteryl substituted groups. AFM investigation indicated some aggregated domains with the averaged height about 3.6 ± 0.2 nm appeared. This suggested an organized structure of double molecules may be fabricated in the transferred LB films. The present results have demonstrated that the interfacial properties of luminol derivative can be modified by changing proper substituted groups of luminol, which show potential application in functional material fields such as ECL sensor.