Sodium is one of the most common metal ions in biology; however, DNA-based sodium probes have only been reported recently. A Na+ -specific RNA-cleaving DNAzyme named NaA43 is active with Na+ alone. In this work, we were using Co(NH3 )6 3+ as the intended metal cofactor for in vitro selection, but obtained a mutant of the NaA43 DNAzyme. The mutant was named NaH1, and differs from NaA43 by only two nucleotides. NaA43 has an optimal pH of 7.0, whereas the optimal pH for NaH1 is 6.0. This difference might be due to our selection having been performed at pH 6.0. NaH1 also displays an excellent selectivity for sodium relative to other competing monovalent ions, as well as a fast catalytic rate of (0.11±0.01) min-1 with 50 mm Na+ . At low Na+ concentrations, the selected DNAzyme exhibited a higher cleavage rate than NaA43 and thus a tighter apparent Kd of (12.0±1.6) mm Na+ . Furthermore, the NaH1 DNAzyme was engineered into a fluorescent Na+ biosensor by attaching a fluorophore/quencher pair to the DNAzyme with a detection limit of 223 μm Na+ . Preliminary work on detection of Na+ in serum was demonstrated as well. This study provides a useful mutant that works in a slightly acidic environment, which might be useful for sensing Na+ in acidic in vivo environments.