The invention relates to a preparation method of cholesteryl alpha-glutamate gel. According to the method, the reaction of acid anhydride and alpha-glutamic acid is used for protecting alpha-amino, the intramolecular dehydration is performed to form cyclic glutamate anhydride, then the cyclic glutamate anhydride has esterification reaction with cholesterol to generate alpha-amino protected cholesteryl glutamate, and then hydrazine hydrate is used to remove the protection group anhydride to synthesize the cholesteryl alpha-glutamate. The cholesteryl alpha-glutamate gel prepared by using the method can overcome the problems that the reaction condition for preparing the cholesteryl alpha-glutamate gel by virtue of direct esterification is not easy to control and the yield is low; the gel prepared by the method can effectively improve the biocompatibility and gelling property, and a useful means is provided for preparing a drug carrier and a sensor material.