Biohybrid nanoreactors operating in one-pot cascade reactions were designed by co-immobilization of enzymes in an inorganic layered matrix, namely, layered double hydroxides. These biohybrid systems were devoted to prepare dihydroxyacetone phosphate (DHAP) and phosphorylated sugars through stereoselective C−C bond formation. In the first system, two kinases were exploited for the in situ generation of DHAP. Increasing the complexity, the second nano-bioreactor combined up to four enzymes to lead to d-fructose-6-phosphate from the aldol-catalyzed addition of dihydroxyacetone to d-glyceraldehyde-3-phosphate generated in situ from DHAP. The biohybrid catalyst showed the same reaction rate as that of the free enzymes and was reusable.