The invention discloses a one-pot method for preparing gamma-glutamine compounds. The method comprises the following steps of carrying out water phase amidation on 5-carboxyl ethyl hydantoin and amine compounds so as to form 5-carboxyl ethyl hydantoin amide compounds; after reaction, directly adding thallus containing both hydantoinase and carbamoyl enzyme without extraction and separation, and carrying out enzymatic hydrolysis ring opening reaction by taking the thallus as a catalyst so as to obtain the gamma-glutamine compounds. According to the method, a hydantoin ring is adopted for protecting both the alpha-amino and the alpha-carboxyl of glutamic acid, only the gamma-carboxyl is exposed, so that the position specificity of amidation is guaranteed; by adopting water phase amidation, the medium product does not need to be extracted and separated and can be directly used for enzyme reaction of the next step, in the 'one-pot method' reaction process, the reaction total recovery can be effectively improved, and the water phase reaction also avoids environment contamination caused by an organic solvent; and the enzymatic hydrolysis ring opening reaction avoids repeated protection and deprotection steps in most of the chemical synthesis reactions and reduces side reactions.