In multienzymes cascade reaction, the inter‐enzyme spacing is supposed to be a factor affecting the cascade activity. Here, a simple and efficient Y‐shaped DNA scaffold is assembled using two partially complementary DNA single strands on magnetic microspheres, which is used to coimmobilize glucose oxidase (GOD) and horseradish peroxidase (HRP). As a result, on poly(vinyl acetate) magnetic microspheres (PVAC), GOD/HRP‐DNA@PVAC multienzyme system is obtained, which can locate GOD and HRP accurately and control the inter‐enzyme distance precisely. The distance between GOD and HRP is regulated by changing the length of DNA strand. It showed that the cascade activity is significantly distance‐dependent. Moreover, the inter‐enzyme spacing is not the closer the better, and too short distance would generate steric hindrance between enzymes. The cascade activity reached the maximum value of 967 U mg−1 at 13.6 nm, which is 3.5 times higher than that of free enzymes. This is ascribed to the formation of substrate channeling.