Metal ions play a critical role in the RNA-cleavage reaction by interacting with the scissile phosphate and stabilizing the highly negatively charged transition state. Many metal-dependent DNAzymes have been selected for RNA cleavage. Herein, we report that the Ce13d DNAzyme can use nonmetallic iodine (I2) to cleave a phosphorothioate (PS)-modified substrate. The cleavage yield exceeded 60% for both the Rp and Sp stereoisomers in 10 s, while the yield without the enzyme strand was only ∼10%. The Ce13d cleavage with I2 also required Na+, consistent with the property of Ce13d and confirming the similar role of I2 as a metal ion. Ce13d had the highest yield among eight tested DNAzymes, with the second highest DNAzyme showing only 20% cleavage. The incomplete cleavage was due to competition from desulfurization and isomerization reactions. This DNAzyme was engineered for fluorescence-based I2 detection. With EDTA for masking metal ions, I2 was selectively detected down to 4.7 nM. Oxidation of I- with Fe3+ produced I2 in situ, allowing detection of Fe3+ down to 78 nM. By harnessing nonelectrostatic interactions, such as the I2/sulfur interaction observed here, more nonmetal species might be discovered to assist DNAzyme-based RNA cleavage.