In the production crystallization of glucose, there are special problems at the nucleation stage, which requires seed crystals. The need for them reaches 10–15% of the weight of the solution, reducing the productivity of equipment. In this paper, the results of the studies on the identification and creation of effective seed crystals for the nucleation of anhydrous glucose in the presence of surface active agents (SAA) have been described. The nucleation process was controlled according to a change in the transparency of solutions and microscopy. The following have been tested as seeds: small (< 60 microns) commercial anhydrous glucose crystals and the same crystals wet with propanol; large (> 200 μm) and small (< 60 μm) hydrate glucose crystals wet with propanol. The large and small hydrate glucose crystals preliminarily wet with propanol or another aliphatic alcohol is recognized as the best of the tested seeds. When these crystals were mixed with a supersaturated glucose solution at a temperature of 60°C, they rapidly (within 15–30 min) disintegrated into a lot of uniform tiny particles with a size of 1–5 μm, became crystallization centers and began to grow rapidly in the form of anhydrous glucose. A similar phenomenon was also observed when nucleating with the hydrate crystals wet with propanol and at a temperature below 50°C. Based on the tests, new types of seed crystals and a method for preparing thereof in the form of alcohol suspensions of ground anhydrous glucose crystals and crystals of any sizes of hydrate glucose have been proposed.