The invention discloses trehalose synthase as well as a preparation method and application thereof, and belongs to the field of gene engineering and enzyme engineering. Glutamic acid in the 289th site near the active center of the trehalose synthase of Thermobifida fusca YX is mutated into glycine to obtain a mutant E289G; histidine in the 295th site is mutated into an asparagine mutant H295N; methionine in the 344th site is mutated into a lysine mutant M344K; methionine in the 367th site is mutated into a leucine mutant M367L; double mutation is performed on the basis of H295N to obtain mutants H295N/E289G, H295N/M344K, H295N/M367L and H295N/M344K/M367L. Through the mutants, the effect that the conversion efficiency for trehalose preparation by the trehalose synthase cannot be greatly influenced when certain glucose is contained in a substrate is achieved; relatively high industrial value is realized.