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Enzymes, as elements with catalytic functions, can be rationally designed and multiple assembled to form a composite catalytic system and achieve cascade catalytic functions. Enzyme cascade catalysis could produce various chemical products with high conversion rate in short time. With the development of DNA nanotechnology, assembling enzymes to different nucleic acid-based scaffolds in different spatial organizations could effectively improve the catalytic efficiency of enzymes. Herein, we review the construction and application of nucleic acid-based scaffold systems from the perspective of template assembly in three dimensions. The challenges and future outlooks in the development of enzyme cascades are also discussed. KEY POINTS: • The principles and construction of various nucleic acid scaffolds are summarized • The application of nucleic acid scaffolds in enzyme cascade catalysis is discussed., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)