A three-enzyme cascade reaction was successfully realized in a continuous flow microreactor. The first enzyme (Candida antarctica lipase B, also known as Pseudozyma antarctica lipase B) and the third enzyme (horseradish peroxidase) of the cascade process were immobilized in a mild non-contact manner via ssDNA-ssDNA interaction in discrete zones on the capillary wall, whereas the second enzyme (glucose oxidase) was kept in the mobile phase. The unique combined feature of patterning, possibility of loading and stripping, and modularity in a fused silica microchannel is demonstrated. By changing the distance between the two enzyme patches, the reaction time available for glucose oxidase could be independently and modularly varied. The reusability of the enzymatic microfluidic system was shown by using the hybridization and dehybridization capabilities of DNA as a tool for subsequent enzyme immobilization and removal.