BACKGROUND Dehydroepiandrosterone (DHEA), a steroid hormone drug and a precursor of other steroid hormone drugs, is industrially important. We aimed to establish the co-immobilization of keto reductase and glucose dehydrogenase by a crosslinked enzyme aggregate (co-CLEA) and to determine whether co-CLEAs can improve DHEA yield compared with crude enzymes. RESULTS Heterologous expression plasmids containing genes coding for keto reductase or glucose dehydrogenase were constructed. The size of each of these proteins was determined using SDS-PAGE and purification on a nickel column. After optimizing the expression conditions for these two enzymes, the co-CLEAs were prepared with 80% acetone as a precipitant and 0.005% glutaraldehyde as a crosslinking agent. Compared with the crude enzyme solutions, the co-CLEAs showed better thermal stability and pH stability. After six cycles of use, the co-CLEAs still showed 70% (keto reductase) and 46% (glucose dehydrogenase) of their initial activities. After four reaction cycles, the accumulated production of DHEA using the co-CLEAs reached 183 g L-1. CONCLUSIONS This research provides a possible strategy for the production of clinically useful DHEA through co-immobilization.