The invention discloses whole grain highland barley gluten which comprises the following components in percent by mass: 2 to 6 percent of wheat dietary fiber flour, 3 to 9 percent of waxy wheat flour,0.3 to 0.9 percent of Konjac flour, 4 to 20 percent of whole highland barley flour, 4 to 5 percent of salt, 4 to 4.5 percent of a sweetener, 20 to 25 percent of water, and the balance of wheat meal. In addition, the invention further discloses a preparation method of the whole grain highland barley gluten. The preparation method comprises the steps of mixing, dough making, squeezing and curing, splitting, cooling, mixing and seasoning, packaging, sealing, boxing and the like. The whole grain highland barley gluten prepared through the formula and the preparation method provided by the invention is low in salt and sugar, is not added with any chemical additives, tastes good, and is nutritional and healthy; the preparation method provided by the invention is simple and easy to control, is relatively low in cost, and is easy to achieve large scale promotion and production.