Abstract: An allulose 3-epimerase mutant, a genetically engineered bacterium expressing the mutant, and an immobilized allulose 3-epimerase enzyme and an immobilization method thereof are described. A high-throughput screening method is used to obtain an allulose 3-epimerase mutant efficiently expressed in a fermentation process, which can catalyze efficient conversion of fructose to D-allulose, providing an efficient production path for key enzymes required in a D-allulose production process. Additionally, the allulose 3-epimerase is bonded to an immobilizing resin to prepare an immobilized allulose 3-epimerase enzyme. The immobilized enzyme can be applied to batch or continuous reactions to catalyze efficient conversion of fructose to D-allulose.