In this work, a rigid DNA tetrahedron (TDN) scaffold was synthesized to precisely control the interenzyme distance by randomly anchoring two pairs of horseradish peroxidase (HRP)/glucose oxidase (GOx) at the vertices of TDN, which could not only avoid the drawbacks of poor controllability and biocompatibility from traditional scleroid skeletons, but also overcome the defect of imprecise regulation for interenzyme distance caused by DNA origami. Impressively, by varying the side length of TDN scaffold, the interenzyme distance was precisely regulated, thus, an optimal TDN scaffold with highest catalytic efficiency was acquired and subsequently applied for constructing an ultrasensitive biosensor for DNA detection with a low detection limit of 3 fM. This strategy paved an avenue for developing new reliable scaffold to precisely regulate the catalytic efficiency of enzyme cascade reaction with ultimate applications in bioanalysis and clinical diagnosis.