D-allulose is a novel functional sugar substitute primarily produced from the high-cost substrate D-fructose, using D-allulose 3-epimerase to achieve the conversion. Identifying low-cost raw material alternatives to D-fructose for D-allulose production is crucial for its industrialization. In this study, glucose isomerase and D-allulose 3-epimerase were linked with either flexible or rigid peptide linkers in different sequential orientations, constructing four configurations of fusion proteins. The fusion protein configuration, exhibiting the best catalytic performance, was selected and optimized to catalyze D-allulose from the lower-cost substrate D-glucose. The results showed that fusion proteins AE3G and AS3G possessed both glucose isomerase and D-allulose 3-epimerase activities, converting D-glucose to D-fructose and D-allulose. AE3G, connected with a rigid peptide linker, demonstrated higher activity than AS3G, which connected with a flexible linker. The optimized catalytic conditions for AE3G were temperature of 65℃, Tris-HCl buffer of 50mmol/L, pH of 7.5, Co²⁺ concentration of 0.5mmol/L, Mn²⁺ concentration of 0.5mmol/L, and wet cell concentration of 75g/L. Under these conditions, using 100g/L D-glucose or volume fraction 5% F55 high-fructose corn syrup as substrates, the fusion protein produced 17.2g/L and 12.5g/L D-allulose, respectively, with a mass ratio of D-glucose, D-fructose, and D-allulose in the reaction mixture of 2.5∶2.3∶1.0. This study aimed to verify the feasibility of using fusion proteins to catalyze the conversion of D-glucose to D-allulose, and the results indicated that the equilibrium concentration of D-allulose was higher than reported values, providing a theoretical basis for low-cost D-allulose production.