Objective To develop a method combining enzymatic catalysis and resting-cell biotransformation to produce allitol from low cost substrate d-glucose. Results The recombinant E. coli expressing d-psicose-3-epimerase (DPE), ribitol dehydrogenase (RDH) and formate dehydrogenase (FDH) for allitol production from d-fructose was constructed. The optimizations of the cell catalytic conditions and the cell cultivation conditions were made. Then, 63.4 g allitol L-1 was obtained from 100 g d-fructose L-1 in 4 h catalyzed by the recombinant E. coli cells. In order to decrease the substrate cost, d-glucose was used as the substrate instead of d-fructose and immobilized glucose isomerase was used to convert d-glucose into d-fructose. In order to simplify allitol production process from d-glucose, one-pot reaction using the mixed catalysts was used and the reaction conditions were optimized. Finally, 12.7 g allitol L-1 was obtained from 50 g d-glucose L-1 catalyzed by the mixed catalysts of immobilized glucose isomerase and the recombinant E. coli cells. Conclusions Allitol can be efficiently produced from low cost substrate d-glucose by using the method combining enzymatic catalysis and resting-cell biotransformation, which is the first report.