<p>Developing a DNA nanobooster-constructed aptasensing platform that enables high-fidelity imaging in living biosamples is of importance for detecting disease-correlated marker molecules. Here, we explore a direct-lock near-infrared (NIR) light-controlled strategy under apurinic/apyrimidinic endonuclease 1 (APE1) gating, which is then incorporated with a sensitive auto-reinforcing iterative cascaded DNA nanobooster. In one respect, the sensing system gains forceful bio-targeting proficiency by applying endogenous APE1 in biological media as a sensor gating, followed by implementing a direct-lock NIR light-controlled strategy through the combination of ultraviolet-emitting upconversion nanoparticles and a 7-(Diethylamino)coumarin-4-ylmethyl-group linkage at the apurinic/apyrimidinic site of one DNA strand. In the other aspect, a first-stage hybridization chain reaction (HCR) amplification and a later-stage iterative HCR cycle amplification triggered by a self-propelled Mn²⁺-dependent DNAzyme are integrated to devise a sensitive auto-reinforcing cascade. Upon appointing adenosine triphosphate (ATP)—a small molecule with dysregulated contents in various diseases like cancer—as a model target, our sensor is confirmed to have high sensitivity and exceptional specificity. Crucially, this aptasensing platform showcases high-fidelity imaging capability for low-abundance ATP from living cells to nude mouse bodies, contributing to an efficient tool for bioassays.</p> Graphical abstract <p></p>