Herein, for the first time, we propose that the cleavage activity of DNAzyme is accompanied by the release of hydroxyl ions, which can be used for colorimetric assay. Subsequently, we further construct a colorimetric strategy for lipopolysaccharide (LPS) analysis by using this property. Detailly, DNAzyme is split into two fragments separately modified with aldehyde group and hydroxylamine group, which can be linked together through oxime chemistry and the presence of LPS can prevent the formation of oxime bond. The formed whole DNAzyme can mediate the release of hydroxyl ions serving for colorimetric signal output. Taking LPS as model targets, DNAzyme-based colorimetric assay has been successfully constructed. This work not only provides a colorimetric strategy to analyze DNAzyme activity, but also gives a new insight to enrich the versatility of DNAzymes and to enhance their multifunctionality.