Substrate channeling, in which metabolic intermediate is directly passed from oneenzyme to the next enzymein an enzyme cascade, accelerates the processing of metabolites and improves substrate selectivity. Synthetic design and precise control of channeling outside the cellular environment are of significance in areas such as synthetic biology, synthetic chemistry, and biomedicine. In particular, the precise control of synthetic substrate channeling in response to light is highly important, but remains a major challenge. Herein, we develop a photo-responsive molecule-based synthetic substrate channeling system on DNA origami to regulate enzyme cascade activity. The photo-responsive azobenzene molecules introduced into DNA strands enables reversible switch the position of substrate channeling to selectively activate or inhibit the enzyme cascade activity. Moreover, DNA origami allows precise control of inter-enzyme distance and swinging range of the swing arm to optimize the regulation efficiency. By combiningthe accurate and addressable assembly ability of DNA origami and the clean,rapidand reversible regulation of photo-responsive molecules, this light-driven substrate channeling system is expected to find important applications in synthetic biology and biomedicine.