DNA hydrogels are powerful candidates for stable and sensitive detection of disease-related nucleic acids. However, the ability to accurately detect is the cornerstone of disease diagnosis. To improve the accuracy of DNA hydrogels for detecting targets, we herein reported the design of pH-responsive DNA hydrogels with ratiometric fluorescence. The DNA hydrogels were prepared from the pH-sensitive ZnO-NH and CO-Y-DNA probe assembled by the three complementary strands. With the use of miRNA-21 as the model analyte, the DNA hydrogels were applied to fluorescence ratio detection. Under acidic conditions, the ZnO-NH was decomposed, thereby releasing the CO-Y-DNA probe. Target miRNA-21 hybridized to the CO-Y-DNA probe, causing the change of fluorescence ratio between TAMRA and Cy5 that both modified in the CO-Y-DNA probe. The developed DNA hydrogels exhibited high accuracy and sensitivity with a low detection limit to 83 pM. In addition, the DNA hydrogels showed long-term stability against DNase I and GSH.