Allitol is generally used as a novel functional sweetener with great potential applications in food, medical care and other fields. Based on the Izumoring strategy, a multienzymatic cascade system involved glucose isomerase, D-psicose 3-epimerase and ribitol dehydrogenase was established in Escherichia coli for the production of allitol from cheap substrate D-glucose. The cofactor recycling system was constructed using the formate dehydrogenase to improve the regeneration efficiency of intracellular reduced nicotinamide adenine dinucleotide. To balance the individual expression differences in the multienzymatic cascade system, the fermentation and catalytic conditions of the cell factory were optimized using whole-cell catalytic activity as an indicator. According to the results, the recombinant bacteria could obtain better protein expression was obtained when cells were incubated at 20℃ for 24h with 1.00mmol/L isopropyl-β-D-thiogalactoside. Under catalytic conditions of 40℃ and 50mmol/L Tris-HCl (pH 8.0) with 5.0g/L sodium formate, the recombinant cells could produce 19.33g/L allitol from 25.00g/L D-glucose. The residual amounts of substrate and intermediates were significantly reduced and the yield of allitol was further improved to 21.12g/L through expression system optimization. The whole-cell biotransformation method could provide a theoretical basis for the large-scale synthesis of allitol, accompany with producing allitol from pretreated sugarcane molasses, which could have positive significance in extending the sugar industry chain and increasing the added value of sugar products.