Recently, we reported two classes of Zn2+-dependent DNA-hydrolyzing deoxyribozymes. The class I deoxyribozymes can adopt a secondary structure of either hairpin or stem-loop-stem. The corresponding most active representatives, I-R1 and I-R3, exhibit single-turnover kobs values of ∼0.059 and ∼1.0 min–1 at 37 °C, respectively. Further analysis revealed that I-R3 could perform slow multiple-turnover catalysis with a kcat of ∼0.017 min–1 at 37 °C. In this study, we sought to retrain and optimize the class I deoxyribozymes for robust single- and multiple-turnover cleavage activities. Refined consensus sequences were derived based on the data of in vitro reselection from the degenerate DNA pools. By examining individual candidates, we obtained the I-R1 mutants I-R1a-c with improved single-turnover kobs values of 0.68–0.76 min–1 at 37 °C, over 10 times faster than I-R1. Meanwhile, we further demonstrated that I-R1a–c and I-R3 are thermophilic. As temperature went higher beyond 45 °C, I-R3 cleaved faster with the kobs value reaching its maximum of ∼3.5 min–1 at 54 °C. Using a series of the kobs values of I-R3 from 37 to 54 °C, we calculated the apparent activation energy Ea to be ∼15 ± 3 kcal/mol for the DNA-catalyzed hydrolysis of DNA phosphodiester bond. In addition, we were able to design a simple yet efficient thermal-cycling protocol to boost the effective kcat of I-R3 from 0.017 to 0.50 min–1, which corresponds to an ∼30-fold improvement of the multiple-turnover activity. The data and findings provide insights on the enzymatic robustness of DNA-catalyzed DNA hydrolysis and offer general strategies to study various DNA enzymes.