The development of highly efficient, recyclable, and multifunctional biocatalysts is of great importance for various applications, especially in biosensing. In this study, highly catalytic and recyclable DNAzyme functionalized poly-N-isopropylacrylamide (pNIPAM) microgels are prepared via one-step precipitation polymerization. The pNIPAM/DNAzyme microgels exhibit highly catalytic activities in aqueous solution at room temperature, and become hydrophobic and separable from the reaction mixture at temperature higher than the lower critical solution temperature of pNIPAM, which facilitate the recyclable utilization of these catalysts. Different kinds of DNAzyme functionalized catalytic microgels can be facilely prepared via the one-step synthesis procedure. Two typical catalytic DNA structures, the Mg 2+ -dependent DNAzyme and the hemin-G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme, are chosen as model systems to validate the feasibility. These pNIPAM/DNAzyme microgel catalysts maintain 80% to 91% initial catalytic activity after eight times of catalysis recycling. Furthermore, the pNIPAM microgels by themselves provide additional interfaces to capturing an enzyme, glucose oxidase, which can cascade with the linked HRP mimicking DNAzymes, to form recyclable bi-enzyme cascading system for the sensing of glucose.