In living organisms,most biochemical processes are implemented and mediated by a series of enzymatic reactions in a cascade way.[1] Such cascade reactions could occur specifically and efficiently,relying on the appropriate spatial arrangement of multi-enzyme on a scaffold(e.g.,cytoskeleton or cell membrane)to avoid subsidiary reactions.[2] The key of mimicking such type of reactions in vitro is to build a reliable scaffold capable of locating multi-enzyme with precisely controlled position and distance.Developments of DNA nanotechnology[3] provide a promising way to solve this problem,owing to the amazing properties of DNA nanostructures,such as structural programmability,[4] accurate addressability and site-specific functionalization.[5] Herein,we use a DNA machine[6] as the scaffold to locate two cascade enzymes,and regulate the efficiency of the enzyme cascade reaction in situ by controlling the distance between them dynamically and reversibly.When the DNA machine is in the open state,the distance between GOx and HRP(the two cascade enzymes)is about 18 nm,while the DNA machine is closed by strand-displacement reaction,the distance decreased to about 6 nm.By monitoring the reaction at 410 nm with UV-Vis spectroscopy,we could evaluate the efficiency of enzyme cascade reaction.[7] Compared with the enzyme cascade reaction catalyzed by both free enzymes,the reaction catalyzed by enzyme-functionalized DNA machine in the closed state showed an approximately 88%higher efficiency and this enhancement was attributed to spatial organization of both enzymes on the DNA machine,which provided a much closer diffusion pathway for intermediate.After adding fuel strand,reaction efficiency decreased significantly.By sequential addition of fuel and antifuel strands to the enzyme-functionalized DNA machine,we further verify that the regulation of the enzyme cascade reaction is reversible.