Functional luminol derivative with cholesteryl substituted group has been designed and synthesized from the reaction of the corresponding precursor cholesteryl chloroformate with luminol. It has been found that depending on cholesteryl substituted group, the formed luminol derivative showed different properties, indicating distinct regulation of molecular skeleton. UV and IR data confirmed commonly the formation of imide group as well as cholesteryl segment in molecular structure. Thermal analysis showed that the thermal stability of luminol derivative with cholesteryl segment was different from luminol. The difference of thermal stability is mainly attributed to the formation of imide group and cholesteryl substituent group in molecular structure. The present results have demonstrated that the special properties of luminol derivative can be turned by modifying molecular structure of objective compound with proper substituted groups, which show potential application in functional material fields such as liquid crystal and ECL sensor.