To develop cost-effective, biobased, industrial trehalose production from maltose, an integrated bioprocess for trehalose production and separation from maltose with recombinant trehalose synthase in permeabilized cells is proposed in this study. We have successfully established an efficient production system for recombinant trehalose synthase (TreS) (9234 U/mL) in a 50 L fermentor and efficient processes for separation and purification to achieve comprehensive utilization of the raw material and products. The trehalose conversion rate of 75% at 30 degrees C can be reached with 6% glucose generated as byproduct using maltose syrup (30 wt %) as substrate in the bioreactor system via whole-cell biocatalysis. After two-stage simulated moving bed chromatography (SMB), the trehalose could be successfully separated, crystallized, and identified with a 97.6% purity and 95.9% recovery yield, respectively. The yield of trehalose produced was 0.675 g per gram of maltose consumed in the whole process within 80.5 h.