Biochemical reactions are essential biological processes of living systems, which are also significant in many fields, such as biomedicine, cytobiology, synthetic biology, and chemical biology. However, there remains an everlasting requirement for the improvement of efficiency, rate, and controllability of biochemical reactions. Inspired by biological systems that enable precisely controlled multistep biochemical reactions by spatially arranging reactants in a highly organized manner for improving the cascade reaction rate and efficiency, a lot of strategies have been used to organize reactants and construct artificial biochemical reactions. With the advantages of easy programmability, site addressability, and biocompatibility, DNA self-assembly technology has shown great promise in improving biochemical reactions. In this review, we introduce the biochemical reaction improvement strategies and DNA self-assembly technology as well as summarize the recent advances in the applications of DNA self-assembly technology for improving biochemical reaction performances, including DNA cascade reactions, enzyme cascade reactions, receptor reactions, organelle interactions, and cell-cell interactions. We also present the existing challenges and further perspectives of applying DNA self-assembly technology in biochemical reaction regulation and improvement.