An entropy-driven DNA circuit offers an efficient means of sensitive analyte detection with signal amplification. In this article, we rationally engineered an aptamer-based entropy-driven signal-off DNA circuit for colorimetric detection of small molecules. The proposed signal-off DNA circuit is activated by target small molecule binding to drive the collapse of G-quadruplex DNAzyme, accompanied by the colour change of the detection solution from dark blue to light blue. Entropy-driven recycling hybridization significantly magnified the input signal of the target small molecule. Such an assay enables naked-eye detection of adenosine triphosphate and oxytetracycline at concentrations as low as 0.5 μM and 1 μM respectively. Moreover, when compared with the signal-on DNA circuit, the entropy-driven signal-off DNA circuit for colorimetric detection has two advantages. Firstly, unlike in the signal-on DNA circuit, the unavoidable formation of waste complexes in the absence of a target in the signal-off DNA circuit has no influence on target detection performance as its background signal is only determined by the substrate complex. Secondly, the signal-on DNA circuit cannot distinguish false-positive signals generated by invasive catalysts (e.g., HRP, serum, Fe3O4), while the signal-off DNA circuit can distinguish those signals as undesired signals. Overall, the signal-off DNA circuit affords a novel strategy for sensitive and accurate detection of small molecules.