An electrochemical biosensor was developed for the detection of adenosine triphosphate (ATP) based on target-induced conformation switching and nicking endonuclease (NEase)-assisted signal amplification. The electrochemical biosensor was constructed by base pairing and target recognition. After capture DNA hybridized with the gold electrode, a significant current of Methylene Blue (MB) was obtained by differential pulse voltammetry. In the presence of ATP, the hairpin DNA formed a G-quadruplex structure due to the specific recognition between hairpin DNA and ATP. Then the exposed part of the target–aptamer complex hybridized with the 3′-terminus of capture DNA to form a specific nicking site for Nb.BbvCI, which led to NEase-assisted target–aptamer complex recycling. The released target–aptamer complex hybridized with the remaining capture DNA. Nb.BbvCI-assisted target–aptamer complex recycling caused the continuous cleavage of capture DNA with MB at its 5′-terminus, resulting in release of a certain amount of DNA fragment labeled with MB. Then the current value decreased significantly. The reduced current showed a linear range from 10 nM to 1 μM with a limit of detection as low as 3.4 nM. Furthermore, the proposed strategy can be used for the detection of similar substances.