The invention discloses a preparation process of N-carbamylglutamic acid. The invention relates to the technical field of biochemical materials. The preparation process comprises glutamic acid, urea,a catalyst, concentrated hydrochloric acid, distilled water and a plurality of reaction tools. Meanwhile, the preparation efficiency of the N-carbamylglutamic acid is gradually improved by arranging areaction box with a heating device, an electric heating sleeve, a vacuum drying box, a circulating water vacuum pump, an ultralow-temperature refrigerator, a pulverizer, an electronic balance, a PH meter, a centrifugal machine, a high-power electric stirrer, an ultrapure water system and the like, so that the preparation efficiency of the N-carbamylglutamic acid is greatly improved; and in addition, the preparation process can be further suitable for factory-like large-scale processing, and the cost for preparing the N-carbamylglutamic acid is reduced.